A request by the BBC for the Government to lift an injunction banning the broadcast of a Panorama special programme about the alleged plot was turned down by the Government last night.
The programme, a result of "many months" investigation, was to have been broadcast last night, according to a BBC statement.
A report in the New York Times alleging an assasination attempt prompted the call by the BBC for the injunction to be lifted. The paper said agents placed a bomb under a car believed to be the Colonel's.
But a BBC spokesman refused to confirm reports last night that it intended to challenge the injunction in the High Court today.
A statement said: "We are giving urgent consideration to our next step in the belief that the public interest now requires full examination of the allegations made by Mr Shayler."
The BBC insisted that it had made every effort to ensure that nothing in its programme would threaten the safety of security personnel.
The move came as the Government flatly denied claims that British agents carried out an assassination attempt on the Libyan leader.
Yesterday a spokesman for the Foreign Office, the department with responsibility for MI6, the overseas secret intelligence service (SIS), said there had never been such a plot.
"The central claim that there has been a plot to kill Colonel Gaddafi is rubbish," said a spokesman.
"It's inconceivable that in a non-wartime situation that the Government would authorise the SIS to bump off a foreign leader.
"In theory, the SIS can carry out assassinations but only at the express request of the Foreign Secretary. We are really talking only a wartime situation."
The spokesman refused to say how many assassinations had been carried out by the SIS in the last 20 years. But it is understood that Colonel Gaddafi and his Libyan regime are targets of ongoing intelligence operations by MI6.
The Foreign Secretary in February 1996 - the man who would have had to give permission for any assassination - was Malcolm Rifkind, who lost his seat at the last election. Yesterday he was unavailable for comment.
Mr Shayler, who is in jail in Paris awaiting extradition to Britain for an alleged breach of the Official Secrets Act, has made a string of claims. He left MI5 last year to make a series of revelations in a Sunday newspaper.
Last night Mr Shayler's lawyer, John Wadham, refused to say whether his client had made the allegations about Gaddafi. "If it is the case that the security services were involved in a plot to assassinate the leader of another country then that is a matter that is in the public interest and the public have a right to know," said Mr Wadham.