Theresa May has hinted that she could block a second independence referendum, on the eve of her party’s annual conference in Birmingham.
In her strongest comments yet on the issue of another vote, the Prime Minister said that her response would be “overwhelmingly no”.
Downing Street could veto a second poll, as the legal right to hold one resides at Westminster.
But senior figures within No 10 fear that would risk increasing support for independence.
First Minister Nicola Sturgeon has said that another vote is "highly likely" in the wake of the shock Brexit result.
In an interview with BBC Scotland Mrs May said that the key issue was not whether there could be a second referendum but whether there should be one, and to that her answer was "overwhelmingly no".
She added: "I continue to believe that Scotland is better off as part of the United Kingdom, and I believe passionately in the strong union that there has been between Scotland and the rest of the United Kingdom for so long.
"Our history, the economic ties we have - I want Scotland to remain part of the UK.
"People talk about what's going to happen in terms of leaving the European Union for Scotland's trade - actually the economic importance of Scotland being part of the UK far overwhelms that."
In the interview she also reiterated her position that Scottish Government would be "fully engaged" in Brexit talks, but that her government would take the lead.
She added that she would engage with Holyrood "on the issues that particularly matter to Scotland".
The Tory conference, which Mrs May will attend as leader for the first time this weekend, is expected to be dominated by the issue of Europe.
Mrs May is already under pressure after one of the UK’s largest car manufacturers, Nissan, demanded ministerial guarantees on the effect of leaving the EU.
The firm employs almost 7,000 people in the UK.
Earlier this week International Trade Secretary Liam Fox gave what was widely seen an indication that the UK was headed towards a ‘hard Brexit’, prioritising border control over membership of the single market.
A former Tory minister branded him “almost delusional” for believing that the UK could get a better deal from the EU after Brexit.
Former business minister Anna Soubry also called on Mrs May to come up with a "plan" for the UK's exit.
She said she was worried that "all we are hearing from is people like Liam Fox, who are talking in a way which at times concerns me as almost delusional.”
“I mean the idea we’re going to get a better deal from Europe is nonsense.”
A former top European judge has accused Mrs May’s government of a “great deal of ignorance” over its handling of its Brexit position.
At a conference organised by market analysis firm MLex, Sir David Edward also accused Dr Fox of not understanding trade law.
Asked about concerns over possible new tariffs on UK trade, Dr Fox said: "Who does it harm more if we end up in a new tariff environment? Does it harm more those who sell more to the UK, or the UK?
“It's in everybody's interests that, as we move forward, that we have at least as free a trading environment as we have today."
A UK Government spokesman said: "We are determined to make sure that the UK remains the best place in Europe to run and grow a business, whether it's one operating at home or abroad.
"We are not going to provide a running commentary on every twist and turn of these negotiations. You don't start a negotiation by telling the people you are negotiating with exactly what you plan to do. That approach won't help us get the best deal for Britain."
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