The SNP's inability to rule out a second bid for independence is a bigger uncertainty for Scotland than the EU referendum, David Mundell has said.
The Scottish Secretary insisted June's vote on Britain's membership of the EU should not be a "re-run" of the 2014 independence referendum.
He told MPs that Nicola Sturgeon must rule out the prospect of a second independence referendum if she is returned as First Minister, adding SNP Westminster leader Angus Robertson must also stop "obsessing" about the idea.
Prime Minister David Cameron has warned quitting the EU could lead to the "disintegration" of the United Kingdom.
Ms Sturgeon believes there will be an ''almost certain'' drive for another independence referendum north of the border if Scotland votes to stay in the EU but England votes to leave.
Speaking in the Commons, Mr Mundell replied to a series of SNP questions about the EU referendum by referring to a second independence vote.
After Patricia Gibson (North Ayrshire and Arran) urged the UK Government to make the positive case for staying in the EU, Mr Mundell said: "You and your colleagues repeatedly call for a positive campaign for Scotland to remain in the EU but all we hear about from them is process and calls for a second referendum.
"I call on them to disregard that approach and actually start setting out the positive case themselves."
He added to Gavin Newlands (Paisley and Renfrewshire North): "What I believe is the biggest issue that faces Scotland currently is the uncertainty over the Scottish Government's inability to rule out a second independence referendum, which they could quite easily do.
READ MORE: SNP MPs to launch barrage of Commons questions about Scotland and the EU
"I look forward to the First Minister, if she is re-elected to that post, setting out clearly that we aren't going to have a second independence referendum."
SNP chief whip Mike Weir (Angus) said he wondered if Mr Mundell's "mission is to antagonise as many Scots as possible before the (EU) referendum".
Conservative Kevin Foster asked for Mr Mundell's assessment of last week's Scottish Parliament election results.
Mr Mundell replied: "There was one clear message from last week's Scottish Parliament elections - the people of Scotland don't want another referendum and I hope the First Minister has heard that message loud and clear.
"The EU referendum is about the UK's membership of the EU, it is not a re-run of the Scottish independence referendum."
Shadow Scottish secretary Ian Murray said the SNP is "desperate for any excuse to trigger another independence referendum".
Mr Robertson said Scottish voters have returned a "pro-European SNP government" ahead of June's referendum, adding: "On the powerful case for remaining in the EU, will the UK Government concentrate please on making a positive and inspiring case to stay rather than re-warming endless scare stories?"
Mr Mundell replied: "I think I've made it clear to you that that's exactly what my intention is, and perhaps you could undertake today to stop obsessing about process and a second Scottish independence referendum and concentrate yourself entirely on the positive reasons for Scotland remaining in the EU."
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