Downing Street has shot down suggestions a second independence referendum could be held next year.
No 10 said that Theresa May believed Scots had made clear their views on the Union only a “couple of years ago”.
A furious row erupted at the weekend following reports that Scottish ministers were gearing up for a possible 2017 vote.
A spokesman for the First Minister denied that any decision had been made and said that every option would be explored to protect Scotland's interests in the wake of the shock Brexit vote.
But he added that there was “nothing secret about the SNP's intentions”.
Opposition parties reacted by urging First Minister Nicola Sturgeon to concentrate on her "day job" instead of independence.
Ms Sturgeon warned that another referendum was “on the table” in the immediate aftermath of the UK's vote to leave the European Union.
Within days a series of opinion polls appeared to show a significant spike in support for an independent Scotland.
But last month another poll suggested that the proportion of Scots who back independence remains substantially unchanged from 2014.
The legal right to order a binding referendum remains at Westminster.
David Cameron’s government gave Holyrood the power, on a temporary basis, to hold the 2014 vote.
However, there is no guarantee that Mrs May's administration will do the same.
Asked if the Prime Minister was minded to lend Holyrood the ability, Mrs May’s official spokeswoman said: “There was a referendum in 2014 that was legal, decisive and fair.”
She added: “The Scottish people were given the opportunity as recently as a couple of years ago to have their say on Scotland’s place in the Union and the PM has been very clear.
"She opened her statement when she took office in Downing Street underlining the importance that she attaches to the UK and working to deliver in the interests of all people across the UK”.
A motion from the SNP's Edinburgh West branch, due to be debated at the party's autumn conference in Glasgow, states that if no other way can be found of protecting Scotland's EU membership, then preparations for a second independence referendum should start.
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