The Northern Ireland Office has described Martin McGuinness's call for the secretary of state to step down ahead of the EU referendum as "ludicrous".
The deputy first minister made the call following Theresa Villiers's weekend announcement that she would join four other members of David Cameron's cabinet in campaigning for the UK to leave the EU.
Ms Villiers has made no secret of her euroscepticism but her support for the 'Out' campaign is likely to bring the secretary of state into conflict with political parties in the north who are opposed to Britain's exit, known as 'Brexit'.
Sinn Féin, the SDLP and Alliance have made it clear that they will campaign for an 'in' vote.
Of the major Stormont parties only the DUP has so far declared itself eurosceptic. The Ulster Unionist Party's executive will meet next week to agree its policy.
Mr McGuinness yesterday went further than SDLP leader Colum Eastwood, who at the weekend said Ms Villiers should resign if a majority of people in the north chose to remain within the EU.
The Sinn Fein MLA voiced concern that the Conservative MP was not in tune with public opinion in Northern Ireland and called on her to quit.
He said a majority of people in the north placed "great value on our membership of the European Union" and wished for that relationship continue.
"It's odd that Theresa Villiers should now be advocating withdrawing from the EU when its benefits for the north are quite obvious.
"It's not surprising, however, given the fact that she is not elected by and does not represent the people of the north that she should be so cut off from public opinion."
Prime Minister David Cameron yesterday defended the secretary of state's position, saying she "does an excellent job".
"She is exercising her ability to reach a personal decision and to campaign for Britain to leave the EU and that's absolutely right she is able to do that," he said.
An NIO spokesman also said Mr Cameron had made it clear that cabinet ministers would be free to campaign in a personal capacity.
"The secretary of state remains totally focused on her ongoing and determined efforts to build a brighter, more secure future for Northern Ireland," he said.
"To suggest otherwise is ludicrous."
Meanwhile, UUP leader Mike Nesbitt dismissed suggestions that he was dithering over his party's EU stance.
He said the party wanted to "listen to people", including the business community and the voluntary and community sectors.
While not joining the calls for Ms Villiers to resign, Mr Nesbitt said the secretary of state needed to keep her cabinet and parliamentary roles separate.
"She is secretary of state and I would expect her to respect the Chinese wall between her position as secretary of state and as a member of the Conservative party," he said.
