Israeli military police are investigating whether an air strike which killed 21 members of the same family sheltering in a building during the Army's Gaza offensive in 2008-9 was authorised by a senior brigade commander who had been warned of the danger to civilians.

The new turn in the enquiry has cast a fresh spotlight on what is widely thought to be the worst single incident involving civilian casualties during Operation Cast Lead, the missile attack on a building in the Zeitoun district of Gaza City, where around 100 members of the extended Samouni family were taking refuge on the morning of 5 January, 2009.

The missile attack, which also injured 19 people, came early in the ground offensive. According to many Palestinian witnesses, it came after troops in the Givati brigade ordered dozens of family members, including women and children, to move to the building the previous day.

It also coincides with evidence that the attack followed photographs from an aerial drone of men collecting firewood outside the building, including boards from a small structure next to it, which was interpreted by Givati brigade commanders as indicating they were carrying a rocket-propelled grenade launcher. While the first missile – thought to have been fired from a drone – was aimed at the group of men, injuring a few, at least two more landed on the building itself after they had hurried back inside. Interviews with soldiers who were in the area at the time, carried out by Amira Hass of the Israeli daily Haaretz and the Israeli veterans' and human rights group Breaking The Silence, have helped to cast fresh light on just what happened on the morning in question.

Part of the military police investigation is now expected to focus on whether senior officers, including the Brigade Commander, Col. Ilan Malka, were aware of the civilian presence at the location or in the immediate area when he authorised the strike.

The military did not comment yesterday on specific Israeli media reports that airforce officers had already testified to the Samouni investigation that Col Malka had been warned that there could be civilians in the area. Col Malka has reportedly denied that he had any warning of a civilian presence.

The investigation may throw up renewed questions about whether rules of engagement in force during the Operation were too permissive.

According to the Israeli human rights agency B'tselem, 1390 Palestinians were killed during the operation, of whom 759 "did not take part in hostilities". The inquiry may also call into question whether the use of surveillance technology, including imaging from the air, is sufficiently clear to justify such attacks, particularly when not augmented by reliable human intelligence.

The existence of the investigation has already been seen as a potential, if partial, vindication of the UN enquiry under South African judge Richard Goldstone, which severely criticised the military's conduct in the Samouni case, as in many others.

Israel refused to cooperate with the enquiry and has severely criticised it since the judge's report.

The military's Judge Advocate General, Maj. Gen. Avichai Mendelblit ordered almost 50 investigations arising from the operation, though so far only three soldiers have been convicted, one for stealing and using a Palestinian's credit card and two for forcing an 11-year-old boy to open bags which could have contained explosives. Another soldier has been indicted for the fatal shooting of a person in the Gaza village of Juhr Al Dik.

In a report on five initial investigations he ordered after Operation Cast Lead, the military's Chief of Staff, Gabi Ashkenazy, said: "The IDF operated in accordance with moral values and international laws of war... and made an enormous effort to focus its fire only against the terrorists, whilst doing the utmost to avoid harming uninvolved civilians." Ashkenazy's comments came before the Goldstone report appeared.

One of the five initial investigations, under Israel Defence Forces Col. Tamir Yadi, specifically covered "claims regarding incidents in which many uninvolved civilians were harmed" and reportedly did not conclude that there had been anything unusual about the Samouni strike. This was despite graphic and largely consistent accounts by numerous Palestinian witnesses to human rights organisations, Israeli and international media, including The Independent, of the strike on the building. These said that, with those in the building cold, hungry and thirsty, a few men had left the building on the freezing early morning of 5 January in order to find wood to make a fire to make tea and to bake bread, but also to urge another relative nearby to join them in what they thought was a safe refuge. They are said to have regarded the nearby presence of soldiers as a protection.

The IDF declined to confirm a report that Yoav Galant, the outgoing head of Southern Command and the new Chief of Staff Designate, had opposed the military police investigation on the Samouni case.

Breaking the Silence confirmed yesterday that soldiers who had spoken about operations in Zeitoun during the 2008-9 offensive had been convinced that a militant Palestinian RPG squad had been operating in the area, apparently on the basis of the same – incorrect – information that led to the air strike.

That was the information they had been given over the radio by the war room, at a time close to when the strike occurred. Indeed, when a young woman, whose husband had been killed in the attack, subsequently arrrived with her injured baby daughter and her brother-in-law at a house occupied by troops, soldiers simply assumed that they had been the victims of a misfired RPG attack which had been intended for the house they were occupying, instead of a missile attack on the Wael Samouni building, of which they were unaware. The house was one of several taken over by troops during the Zeitoun operation. In all other respects they corroborated the detailed recollection of Maysa Samouni, who did indeed arrive at a house occupied by soldiers and whose injured daughter – who had lost three fingers – was given first aid by soldiers.

Ms Hass' reconstruction, amplifying previous testimony by witnesses from the Samouni family, describes how on the morning of 4 January, force commanders – who are not among those to have talked about the day – ordered dozens of family members to leave the three storey house of Talal Samouni which had been turned into a military position.

They were told to assemble in the one-storey house of Wael Samouni, about 30 metres to the south east.

Ms Hass, who has also interviewed dozens of Samouni witnesses, says the fact that there had been elderly people, women and children were already in the group assembled there and that they had been ordered by the soldiers to go to the building, was a guarantee no harm would come to them. In the event, women and children were among those killed.

Among the several children and young adults orphaned in the blasts was Mona Samouni, now 12, who saw both her parents die at her side.

One of the questions which the current military police investigation will presumably have to decide is how it was – even if Col Malka was not specifically warned that civilians were present before the attack was authorised – that he did not know: why the war room from which the Givati operation was being run was not told the previous day that unarmed civilians, including women and children, had been ordered to move to Wael Samouni's house.

Yesterday, the military would only say that the Samouni attack was "the subject of a military police investigation".