Alex Salmond has warned Yes supporters that Thursday could be their last chance for a generation to secure an independent Scotland .
But the SNP leader also refused to tie his own hands or those of his successors, leaving the door open for another referendum within just a few years.
He repeatedly said that his stance was merely "my view" and "my opinion" as he spoke of not asking the question again for a "political generation".
Many within the wider Yes movement are keen to hold a second vote if they fall just shy of an overall majority next week.
They argue that support for independence has risen considerably since two years ago, when polls suggest it was backed by just one in three of the population.
Many believe that another push could guarantee separation from the UK - especially if David Cameron wins a second term in Downing Street next year.
Mr Salmond's deputy Nicola Sturgeon has previously suggested a generation could be as little as 15 years.
The SNP leader made the comments as he was interviewed on the BBC's Andrew Marr programme.
The First Minister was asked if he would pledge not to "bring another referendum if you don't win this one". He replied: "That's my view. In my view this is a once-in-a-generation, perhaps even once in a lifetime opportunity for Scotland."
He added: "Harold Wilson famously (said) one vote is enough in a referendum but we're not aiming to win by one vote, we're aiming to achieve a substantial majority if we can.
"If you remember that previous constitutional referendum in Scotland - there was one in 1979 and then the next one was 1997. That's what I mean by a political generation."
There is international precedent for having two separate votes in a relatively short period of time. Quebec held an independence referendum in 1980 and then another in 1995. In the first roughly one-third of voters backed independence - but in the second Quebec came within a whisker of separating from Canada.
On the same programme, Better Together leader Alistair Darling said there was "no way back" from a vote for independence. He said he had always felt "this race would go to the wire" and added that large numbers of voters were "yet to make their minds up".
He added: "It's not surprising. It's the biggest single decision we will ever take.
"If we vote to leave the UK on Thursday then there's no going back. It's not like an election where you can change your mind if things don't work out. If things go wrong this time, we've already voted - we're leaving.
"There is no way back, which is why in the next five days we will be targeting the 500,000 or so voters who have still got to make their minds up, because the decision is that critical."
The Labour former Chancellor said he personally had felt "menaced" in the campaign and had been shouted down simply for appearing at an event.
He said: "I've been involved in political campaigning for about 35 years and I've never seen anything like this before.
"I think the majority of people, the quiet majority who are beginning to speak out, are not prepared to be bullied into accepting there's only one side here."
Mr Darling insisted there would be a deal between the pro-Union parties on new powers for the Scottish Parliament in the event of a No vote because the differences between their positions were "very small".
He said: "It's a clear choice now. We can have faster, safer and better change, reform, so that we can strengthen the health service, we can safeguard jobs, but you don't have the risks that come with independence - to jobs, to the funding of pensions and the health service, the uncertainty about currency."
A Better Together spokeswoman said: "The idea of another two-and-a-half year referendum campaign will horrify the vast majority of Scots."
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