Boris Johnson has rejected the idea that a vote to leave the EU could trigger a better deal from Brussels and a second referendum - insisting he believes "out is out".
The Mayor of London was criticised by David Cameron after appearing to publicly endorse a fresh national ballot when he declared his intention to break ranks and campaign for a "leave" vote.
The Prime Minister returned to the offensive on Friday, calling the idea a "complete fiction" when it was floated by former Tory leader Michael Howard.
It is seen by some pushing for a Brexit as a powerful weapon to win over voters leaning towards wanting to take Britain out of the EU but unsure of the long-term consequences.
In his announcement last week, Mr Johnson wrote in his Telegraph column that "EU history shows that they only really listen to a population when it says 'no' ".
But he told The Times that he was not in favour of using a popular rejection of EU membership as leverage to secure further reforms and concessions from Brussels.
"Out is out," he said. "What I want is to get out and then negotiate a series of trade arrangements around the world."
Asked if he was ruling out a second vote, he said: "I don't think it would be necessary."
The clarification will be welcomed by Mr Cameron, who is on a tour of the UK promoting the case for continued membership amid a swirl of attacks from senior Eurosceptic colleagues.
In the latest blow, Lord Howard said the PM's renegotiation of the UK's membership had "met with failure" and urged voters to back withdrawal.
And Government minister Andrea Leadsom, who is campaigning for the UK to quit, dismissed as "unlikely" Chancellor George Osborne's assessment that leaving the EU would cause a "profound economic shock" for Britain.
Mr Cameron insists he would respond to a Leave vote by immediately triggering Article 50 of the Lisbon Treaty, which sets out a two-year process for negotiating terms for withdrawal.
But Lord Howard suggested he could pause the process by up to a month for EU leaders to come back with a better deal for Britain.
The peer used an article in the Daily Telegraph to argue that the UK should vote for Brexit to "shake Europe's leaders out of their complacency", arguing there would be "a significant chance that they would ask us to think again".
Asked about Lord Howard's comments during a visit to an aviation factory near Cardiff, the PM said: "This idea that there is some third way - as some are suggesting - between in and out, that we vote out in order to have another renegotiation, another referendum, I think that is a complete fiction.
"It's a very simple question on the ballot paper - you either remain in the European Union or you leave the European Union. I think people really do need understand that it's a single decision, it's a final decision, and there are only two choices."
Mr Osborne, in Shanghai for a summit of G20 finance ministers, said global economic instability meant it was "the very worst time for Britain to take the enormous economic gamble of leaving the European Union.
"You've seen the value of the pound fall and it reminds us all this isn't some political parlour game it's about people's jobs and livelihoods and their living standards," he told the BBC.
"What I owe the country as a chancellor is a serious, sober and principled analysis of what the country faces if we leave the EU."
He went on: "In my judgement as Chancellor, leaving the EU would represent a profound economic shock for our country for all of us, and I'm going to do everything I can to prevent that happening."
He said the party was "mature enough" to handle disagreement over Europe.
Unconfirmed reports suggested that the world's most powerful economies may signal their concern over the prospect of Brexit with a statement in the official communique issued at the end of the G20 meeting on Saturday.