The First Minister has said David Cameron’s speech on European Union membership "completely changes the nature of the debate in Scotland".
During his speech on Wednesday, the Prime Minister promised an in/out referendum on the UK’s membership of the EU by the end of 2017 if the Conservatives win the next general election.
He said the Conservative manifesto for the 2015 general election will ask for a mandate to negotiate a "new settlement" for Britain in Europe, which will be put to voters in a referendum within the first half of the five-year Parliament.
But the Prime Minister said he will campaign "with all my heart and soul" for Britain to stay in the EU when the referendum comes.
He also warned voters that if the UK did decide to leave, it would be "a one-way ticket, not a return".
After the talk to a business audience in the City of London, Alex Salmond accused Mr Cameron of giving a “fundamentally confused” speech, saying he was trying to keep everyone happy.
He said: "This was a fundamentally confused speech by the Prime Minister which is painfully short on detail.
"On the one hand he is trying to appease the Eurosceptics on his own backbenches and on the other he is trying to appear as a European reformer. He is trying to ride two horses at the same time and it is inevitable he will fall off before long.
"This completely changes the nature of the debate in Scotland. The Westminster parties have consistently claimed that a referendum on Scotland’s independence causes uncertainty.
"It is now clear the persistent undercurrent of Tory Euroscepticism poses the biggest threat to Scotland's position in the EU and has now helped to hole below the waterline the baseless scaremongering of Alistair Darling and the rest of the No campaign."
Mr Salmond plans on keeping Scotland in the EU if the country becomes independent.
On Wednesday, Mr Cameron called for a new EU treaty to reshape the 27-nation bloc, resolve the problems of the eurozone, allow the transfer of powers back from Brussels to national governments and make Europe's economy more competitive and its institutions more flexible and democratically accountable.
Mr Cameron said it was his "strong preference" to enact these changes for the whole EU, not just Britain alone.
But if other member states are unwilling to go ahead with a new treaty, Mr Cameron said he was ready to renegotiate the UK's position to achieve a settlement "in which Britain can be more comfortable and all our countries can thrive".
Standing in front of a backdrop with the slogan "Britain and Europe", Mr Cameron said: "The next Conservative manifesto in 2015 will ask for a mandate from the British people for a Conservative government to negotiate a new settlement with our European partners in the next Parliament.
"It will be a relationship with the single market at its heart.
"And when we have negotiated that new settlement, we will give the British people a referendum with a very simple in or out choice. To stay in the EU on these new terms or come out altogether. It will be an in/out referendum.
"Legislation will be drafted before the next election. And if a Conservative Government is elected we will introduce the enabling legislation immediately and pass it by the end of that year.
"And we will complete this negotiation and hold this referendum within the first half of the next Parliament.
"It is time for the British people to have their say. It is time to settle this European question in British politics."
Scottish Conservative deputy leader Jackson Carlaw contrasted Mr Salmond's stance on the EU referendum with the Scottish Government's decision to hold a vote on independence.
"On one hand he says the people of Scotland should be allowed an in-out vote when it comes to being part of the UK, but that people of the UK should be denied a similar vote on our future within the EU," Mr Carlaw said.
"We recognise that people have concerns about the current arrangement between the UK and our European partners, and it's only right they get to cast their vote on what that future relationship should be. With Alex Salmond, all we get is uncertainty and assertion.
"He has finally conceded that, under independence, Scotland would not just walk into the EU without negotiation, but has refused to say what concessions would have to be made.
"We have no fear of putting it to the people of Scotland to choose their preferred relationship within the EU, while Alex Salmond and the SNP have no credible proposals on a separate Scotland's position within the EU and certainly no plans to consult the voters."
Scottish Liberal Democrat leader Willie Rennie accused both the Conservatives and SNP of promoting "grinding uncertainty".
"Our priority should be to build a stronger economy in a fairer society but the Conservatives obsess about Europe and the SNP about independence," he said.
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