Two-thirds of Conservative MPs now support Britain’s exit from the European Union, despite David Cameron’s clear preference for staying in, according to senior sources within the party.

Key figures in Tory high command say analysis of public statements and private views expressed by their 330 MPs shows that at least 210 now believe that the UK would be better off “out”.

The surge in support within the parliamentary party for leaving will greatly encourage “out” campaigners, who believe many people will take their lead from local MPs when they decide which way to vote. However, party managers say the total number of Tory MPs who will join the campaign to leave could turn out to be significantly fewer – around 110 – if in the next few months opinion polls begin to point towards a close result or a win for the pro-EU side.

“Certainly at least two-thirds want to leave as it stands,” said a senior party figure. “But if things are very tight some will be bought off by offers of patronage and will be reluctant to take a different line to the prime minister. Plenty will not want their careers blighted by being on the wrong side of such an important debate.” The Observer has also been told that soundings taken by MPs show the “vast majority” of grassroots activists now want to quit the EU – and that most will not be swayed by whatever deal Cameron achieves in his attempt to renegotiate UK membership.

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Last week Cameron, in effect, conceded that his party was split from top to bottom over Europe when he agreed that members of his government, including cabinet ministers, would be allowed to speak out against the official line during the campaign, which is expected to be later this year.

While the holders of the top offices of state – including the chancellor, George Osborne, the foreign secretary, Philip Hammond and the home secretary, Theresa May – are likely to back staying in, other senior ministers, including the work and pensions secretary, Iain Duncan Smith, the leader of the House of Commons, Chris Grayling, and the Northern Ireland secretary, Theresa Villiers, want to campaign to leave.

The spotlight will inevitably now turn to Boris Johnson, who attends cabinet in his role as mayor of London and sees himself as a future leader of the party. A longstanding critic of the EU, Johnson has yet to indicate whether he will campaign to stay in or leave.

The ability of Villiers to remain as Northern Ireland secretary if she sides with the “out” campaign was being called into question on Saturday night as opposition MPs said leaving the EU would not be in Northern Ireland’s interests, could harm the peace process and damage Northern Ireland’s economy. The Liberal Democrat MEP Catherine Bearder, who speaks for her party on the referendum campaign, said Villiers should stand down, saying a Brexit would not be compatible with her role. “It would be highly inappropriate for Theresa Villiers to remain in her post while campaigning to leave the EU,” Bearder said. “Leaving Europe would risk stoking sectarian tensions and undoing years of peace-building, much of it funded through EU peace programmes.

“It would also fundamentally transform the UK’s relationship with the Republic of Ireland and put at risk the open land border we currently share. Government ministers should not be able to campaign for an EU exit if this completely goes against their role and responsibilities.”

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The only Irish nationalist party represented in the House of Commons also questioned whether Villiers could remain in her post. Colum Eastwood, new leader of the Social Democratic and Labour party, said Britain’s departure from the EU could put at risk Northern Ireland’s £1.6bn of trade with the Irish Republic. “While Theresa Villiers is obviously entitled to engage in the internal and long-running Tory battles over Europe, her role as secretary of state for Northern Ireland places upon her a separate responsibility. That role should require her to represent the best interests of people in the north. A Brexit is not in our interest. It is not in the interest of our economy or in the interest of our society,” he said.

“All the evidence, all the major voices in our agricultural and business communities, have warned that a Brexit would devastate the fundamentals of our economy.”

Meanwhile, the EU spokesman of the strongly Eurosceptic Danish People’s party, Kenneth Kristensen Berth, said he did not believe the UK should leave. “I fully understand the British people’s scepticism, but the answer is not to leave now. The answer is to work within the EU framework to slim down the EU cooperation, and that’s a job that will be significantly harder without the British.”

James McGrory, chief campaign spokesman for Britain Stronger In Europe, said: “It shows how isolated Ukip and the Leave campaigns are when even rightwing Eurosceptics in other countries are arguing that Britain should remain in Europe.

“Europe needs reform, but leaving altogether would take us to very extreme fringes of the international community, where even far-right outfits like the Danish People’s party don’t want to be.”