Ed Miliband has flatly ruled out going into coalition with the SNP as he attempted to draw a line under weeks of Tory jibes.
The Labour leader insisted there would be no power-sharing deal with Nicola Sturgeon's party and he would not lead a government that included SNP ministers.
However, he stopped short of dismissing a looser arrangement that could prop up a Labour administration, such as a "confidence and supply" agreement.
The intervention followed growing concern about the damage being inflicted to the party by repeated Conservative attacks on the issue.
With polls suggesting the SNP could win dozens of seats north of the border in May and potentially hold the balance of power, senior Labour figures have repeatedly refused to give direct answers about whether they would consider an alliance.
During furious exchanges at Prime Minister's Questions last week, David Cameron lambasted Mr Miliband as "weak and despicable" for considering a deal with "people who want to break up our country".
A Conservative campaign poster has also depicted Mr Miliband in former SNP leader Alex Salmond's pocket.
But the Labour leader insisted today that a coalition with the nationalists in Westminster "will not happen".
"Labour will not go into coalition government with the SNP. There will be no SNP ministers in any government I lead," ," he said at a town hall-style campaign event in Guiseley, West Yorkshire.
Mr Miliband accused the Tories of "misleading" the public with a "nonsense" scare story.
"I'm not going to spend the next eight weeks speculating about what might or might not happen after the polls have closed," he said.
"It is vital we spend the next eight weeks debating the real choice facing the British people in this election: a choice between a better future for Britain with a Labour government or the failing plan of this Conservative government.
"Our campaign is 100% focused on winning that debate to win the Labour majority government that can bring the change Britain needs.
"The Tories, the party that haven't won a majority for over 20 years, are now running a misleading campaign based on the idea of a Labour-SNP coalition... this idea is nonsense."
Asked whether he was ready to consider a post-election deal to work with the SNP without entering a formal coalition, Mr Miliband said: "I couldn't be clearer about this. I am ruling out a coalition government with the SNP. I am not going to start getting into never-ending speculation about how other parties might vote on a Labour Queen's Speech."
Speaking before Mr Miliband's appearance, Ms Sturgeon said there could be "many things to commend" some kind of tie-up.
"I can't see for the life of me why Labour wouldn't want to contemplate the possibility of working with the SNP to keep the Tories out of office," she said at a lecture to the London School of Economics.
"As long as there are more SNP and Labour MPs than there are Tory MPs, we can lock the Tories out of government, there is no question about that.
"So I won't rule out those other working relationships. In fact, I think they may have many things to commend them."
She said Labour ruling out a formal deal would not "change too much", as the SNP had already said it was highly unlikely.
"What happens after the election will be dictated first and foremost by how people vote in the election and all of us have got to be mindful of not taking people for granted," she added.
A Conservative Party spokesman said: "This changes nothing. Ed Miliband will not rule out a deal with the SNP because he knows it's impossible to become prime minister without being carried into Downing Street in Alex Salmond's pocket.
"There have been over 1,200 votes in this parliament. Vote by vote, bill by bill, issue by issue, Ed Miliband would have to do a deal with the SNP on each and every one of them.
"Who knows what Ed Miliband will sell out to Alex Salmond on: more borrowing, more debt, higher taxes or weaker defences. But one thing's for certain: it's hardworking taxpayers who will pay the price for this chaos."
Meanwhile, a poll for Lord Ashcroft has found Tory support has slipped three points over the past week to 31%.
However, Labour was also down one on 29%, according to the Conservative peer's study.
In an online interview with the BuzzFeed website, Mr Cameron said the Labour leader's refusal to rule out a deal with the SNP was despicable.
"Not ruling out a deal or a pact or support from the Scottish National Party means that the Labour Party is effectively saying 'We're trying to ride to power on the back of a party that wants to break up our country'. I don't think that is acceptable," he said.
"The SNP isn't just any old party - they're a party that thinks the United Kingdom, our country, shouldn't exist. I think it is pretty despicable, frankly, not to say 'There's no dealing with them' ... and he's not saying that."