Downing Street has poured cold water on the prospect of a second referendum if the UK votes to withdraw from the EU on June 23, insisting that "a vote to leave is a vote to leave".
The comment came as David Cameron prepared to face MPs in the House of Commons to lay out his case for staying in the European Union.
The second referendum option had reportedly been floated by London Mayor Boris Johnson - who dramatically came out in favour of Brexit on Sunday - as a means of securing further concessions from the EU.
But the Prime Minister's spokeswoman left no doubt that a Leave vote would trigger the UK's departure by means of Article 50 of the Lisbon Treaty, which provides for a two-year process for negotiating the terms of the departing state's withdrawal and its future relations with the EU.
However, she stopped short of saying that Mr Cameron would immediately trigger Article 50 by informing the European Council of a Leave vote at its summit of EU member-states' leaders scheduled to take place in Brussels on June 23-24.
"If the British people vote to leave, the Government will clearly respect the outcome of that," said the PM's spokeswoman. "They will then launch the process to leave."
Alongside his statement to the House of Commons, the PM is due to release a Government White Paper on the outcome of his renegotiation of British membership, as well as tabling a statutory instrument in Parliament formally setting out the referendum date and the process under which rival official campaign organisations will be designated.
He is likely to get a rough ride in the Commons from Tory backbenchers and half a dozen Cabinet ministers who have already declared they will be voting for Brexit in the national poll.
Sources close to Mr Johnson - who has effectively put himself at the head of the Out campaign by announcing his support - confirmed that the London Mayor and MP is due to attend the statement and will seek to ask a question.
Defence Secretary Michael Fallon denied that Mr Johnson's dramatic move was a "big blow" for those who want to stay in the EU, telling the BBC Radio 4 Today programme the "overwhelming majority" of the Cabinet backed Mr Cameron.
"Obviously you would have liked more support from Boris but he has taken his individual view," said Mr Fallon. "But in the end, when it is all over and the votes have been counted, then you can be sure that the Government will come together again and continue the programme that we got elected on last year."
He also dismissed the idea that Britain could return to a "golden age" where it had full sovereignty without suffering serious consequences.
"The difficulty is this. If you got back to this sort of golden age where our parliament was absolutely sovereign, you would still have the EU next door," Mr Fallon said.
"You would still have the EU taking decisions that affect our trade and our businesses and our way of life and the argument is this - isn't it better to still be there, however frustrating it is, at the table shaping those regulations, leading Europe in the direction you want, protecting your national interests, or should you go out and pursue this illusion of separate sovereignty all on your own without these partnerships and alliances."
Mr Johnson's announcement ended months of speculation about his intentions.
Writing in his weekly column in The Daily Telegraph, the mayor said the referendum offered a "once-in-a-lifetime chance" to deliver "real change" in Britain's relations with her European neighbours.
"There is only one way to get the change we need - and that is to vote to go; because all EU history shows that they only really listen to a population when it says 'no'," he said.
Vote Leave chairman Lord Lawson hailed Mr Johnson's decision.
"I am delighted that he has come out for leaving the EU ... He is a superb campaigner and he is a great asset to the cause," the former chancellor told Today.
Asked for the PM's response, Mr Cameron's spokeswoman said: "The Prime Minister has been clear all along that there will be differing views on differing sides of the argument.
"Our message to everyone - bearing in mind it is people up and down the country that will have a vote in this referendum, rather than just one individual - is that we want Britain to have the best of both worlds."
Mr Johnson's father, Stanley Johnson, a former MEP and chairman of the Environmentalists for Europe group, said it would be a "total travesty" to suggest his son had opted for Brexit as a "careerist" move.
"I cannot think of any more career-ending move than to do what he did yesterday, in the sense that he is leaving the mayoralty in May," said the elder Mr Johnson. "If he wanted to get a nice job in the Cabinet on May 8, this is not the way to do it."
The PM's spokeswoman confirmed that ministers who support EU withdrawal will continue to be allowed to speak from the front bench, but will be required to make clear if they are expressing their "personal view" on Europe, rather than the Government position.
Asked if Mr Cameron was concerned that more than half of his own party's MPs may end up backing Brexit, the spokeswoman said: "We recognise that there are differing views about this issue, that each individual has to weigh up the issues carefully and make their own judgment.
"Because the Prime Minister is delivering a referendum, it means that people up and down the country - not just in the House of Commons - will be able to have their say."