Boris Johnson has overturned an order gagging senior City Hall officials from speaking out in favour of Britain remaining in the EU.
The pro-Brexit London Mayor insisted that he had only learnt on Monday evening of the instruction sent out by his chief of staff Sir Edward Lister, and said he immediately withdrew it, describing it as a "cock-up".
The about-turn comes after Mr Johnson loudly blamed "the agents of Project Fear" for forcing the resignation of John Longworth as director general of the British Chambers of Commerce after he contradicted the organisation's neutral position by personally backing UK withdrawal from the EU.
Sir Edward's email, obtained by the BBC on Monday, stated that "GLA (Greater London Authority) officers, can, when not at work, express personal opinions (which may be contrary to the Mayor's views)."
But it added: "Whilst this is the formal position for you also, I would expect, given your roles, you either to advocate the Mayor's position or otherwise not openly to contradict it".
Mr Johnson's aides initially said the instruction was "in line with that issued by the GLA's statutory officers", but added that the Mayor was "relaxed" about any of his team campaigning for either side in the June 23 referendum in a personal capacity.
But when he was greeted by TV cameras outside his London home on Tuesday morning, Mr Johnson disowned the order absolutely.
"Nobody has been gagged," said the Mayor. "I was only made aware of this edict very late last night and it ceased to be operative as soon as I was made aware of it.
"All I can say is it obviously hasn't been operative because you've got members of my advisory team taking a very different view from me. So they can, so they shall, and with complete impunity too."
Asked whether he had seen Sir Edward's message before it was sent to staff, Mr Johnson said: "I had no knowledge about it. It's a cock-up, I perfectly accept that. It's not something I agree with. My staff and my team have complete freedom to say what they want. They already are and have been for several days."
Quoting former Chinese leader Chairman Mao, he added: "Let a hundred flowers bloom, folks. OK?"
And he said: "As soon as I saw that thing last night it ceased to be operative. Indeed, it had not been operative for several days. It had not been operative at all because they've all been saying exactly what they think. They can do what they want."
Mr Johnson came out in support of the Leave campaign two weeks ago, in conflict with his own party leader David Cameron.
Conservative MP Sir Peter Bottomley said the incident made the Brexit campaign appear like "inconsistent opportunists".
The Worthing West MP, who is backing continued UK membership of the EU, said: "The apparent lifting of this gag has been inevitable.
"The surprise is that the Mayor of London and the Mayor's chief of staff issued the no-alternative order and then failed to withdraw it days ago when the Mayor and other Outers went over the top on the Chamber of Commerce situation.
"My good-natured advice to the Outers is to look in the mirror before heaving a brick into someone's window.
"Cabinet dissenters have their freedom on the EU. What is the weakness of the Out side that caused them to issue the City Hall ban on equivalent freedom? Whether a deliberate error or just a curious mistake, it makes the Outers look like inconsistent opportunists."
The leader of the London Assembly Labour Group, Len Duvall, said the email showed how "panicked" Mr Johnson was by "deep splits even within his own team".
Mr Duvall said: "This is total double standards from the Mayor who complains about David Cameron stifling debate and then tries to do exactly the same thing himself. Despite this climbdown the bullying message he has sent to his advisers is clear - back the Mayor or face the consequences."