At least 12 Libyan rebels are reported to have been killed by a coalition plane enforcing the no-fly zone.
The jet apparently retaliated against a convoy between Brega and Ajdabiya in the north-east after being targeted with an anti-aircraft gun.
Nato is investigating the incident, which took place last night, but it is understood that British pilots were not operating in the area.
News of the casualties came with opposition forces still struggling to make any headway against Muammar Gaddafi's better armed and trained troops.
The dictator's spokesman, Moussa Ibrahim, has dismissed a ceasefire offer by the rebels as "mad", insisting that the government would not withdraw from siege positions around key cities.
Yesterday it emerged that seven civilians, including three girls from the same family, had been killed in another air strike.
Shrapnel was sprayed into nearby houses after a truck carrying ammunition was hit in Zawia el Argobe, according to the BBC.
However, it now appears that the plane involved may have been part of the opposition force, rather than Nato-operated.
The US has said its jets will no longer carry out air strikes from today, although they will be available if assistance is requested by the Nato commander.
That has left the UK, France and Canada responsible for hitting targets on the ground.
Britain has also announced it is providing 2,100 tents for people driven out of their homes by the fighting.
The stocks will be flown from Dubai and distributed by the Libyan Red Crescent, mainly in the Ajdabiya area in the north-east of the country.
Meanwhile, Defence Secretary Liam Fox has said officials are still interviewing Musa Kusa, a close ally of Colonel Gaddafi, who defected from the regime earlier this week.
Asked on the BBC Radio 4 Today programme whether the former intelligence chief would stand trial if evidence was found linking him to the Lockerbie bombing, Dr Fox said: "It's very clear in Britain that our judicial process moves independently from government."
A Foreign Office spokesman said: "Our officials have been in contact with representatives of the Crown Office and Dumfries and Galloway Constabulary in connection with the Lockerbie bombing and will be meeting with them on Monday to discuss the situation concerning Musa Kusa further."