Rebel forces have said the Libyan leader's son Khamis was among more than 30 people dead following an air strike on an operation centre in the western town of Zlitan.
Spokesman for revolutionary militia groups Mohammed Zawawi told AFP that spies operating among Gaddafi's ranks had confirmed the death.
"Overnight there was an aircraft attack by Nato on the Gaddafi operations room in Zlitan and there are around 32 Gaddafi troops killed - one of them is Khamis," he said.
It is the second time this year that there have been claims Khamis has died.
In March, reports the 28-year-old leader of the feared Khamis 32nd Brigade was killed when a pilot crashed his jet into a compound in Tripoli were quickly quashed as rumours.
But there has been no comment from the government or Nato in regards to the latest claims.
He is the second son of the colonel reported to be killed in the Libyan conflict this year.
In April, the leader's son Saif al-Arab Gaddafi and three of his grandchildren were killed by a Nato airstrike on his house.
Col Gaddafi and his wife were said to have been in the building at the time but were unharmed.
The latest reported Nato attacks come as Gaddafi's regime accused the alliance of bombing civilian targets in Zlitan after a mother and her two children were reportedly killed in strikes at 6am on Thursday.
Nato denied it hit the family's home, but confirmed that it did target a command and control facility in the western town.
Zlitan, which lies between the rebel-held city of Misratah and the capital Tripoli, has been home to some of the bloodiest battles in recent months.
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