The Democratic Unionist Party has formally announced its intention to campaign for a Brexit.
The anticipated move from DUP leader and Stormont First Minister Arlene Foster came in the wake of David Cameron's referendum announcement.
Northern Ireland Secretary Theresa Villiers has also joined those advocating an EU exit.
The other three parties in the Stormont Executive - Sinn Fein, the SDLP and the Alliance party - all support the UK staying within the European Union.
The Ulster Unionist Party, which quit the power-sharing coalition administration in Belfast last year, has yet to officially nail its colours to the mast.
Mrs Foster said individual members of her party would be free to take opposing sides in the debate.
"The Democratic Unionist Party has always been Eurosceptic in its outlook," she said.
"At every stage in this European negotiation process we had hoped to see a fundamental change to our relationship with Europe.
"In our view we see nothing in this deal that changes our outlook. Therefore we will on balance recommend a vote to leave the EU.
"Importantly however the decision on whether the United Kingdom should remain in or leave the EU is fundamentally not one for parties but for every individual voter across the nation to determine.
"As every voter has the opportunity to express a view we fully expect that DUP members and voters will hold a range of differing personal views as to what is in the best interests of the United Kingdom. They are fully entitled to do so during what will be a momentous political debate about the direction of travel our nation chooses."
Mrs Foster called for a "positive" campaign.
"There have been far too many scare stories already in this debate," she said.
"People want clarity. They want facts not spin. I hope that, on both sides, the scare stories will now stop as we get down to the serious business of debating our future relationship with Europe."
SDLP leader Colum Eastwood said: "This campaign will be one of the most important votes faced by people in Northern Ireland in decades.
"It is therefore vital that a strong and positive campaign is conducted to remain in Europe."
He added: "Secretary of State Theresa Villiers has today joined the Leave Campaign. As I have told her repeatedly, she does not represent Northern Ireland in this position. She must not attempt to speak on our behalf.
"The simple fact is that the pick and mix of extremists and eccentrics in the Leave Campaign have no Plan B."
Sinn Fein Deputy First Minister Martin McGuinness said the island of Ireland had "benefited greatly" from the EU.
"Benefits of our EU membership can be felt daily across all sections of our society and many organisations and groups have directly benefited from EU funding," he said.
"In particular, our agriculture and agri-food industry have gone from strength to strength with the help of EU Single Farm Payments.
"The EU has also supported key infrastructure projects and boosted our economy through peace and structural funds.
"It has also been a key supporter of the progress made in the peace and political processes over the last two decades.
"Businesses, industry, the agricultural sector and community and voluntary organisations have made it clear that they want to continue to avail of the huge opportunities that exist as a result of our EU membership.
"The future of Ireland, north and south, is within the European Union and Sinn Fein will be campaigning vigorously to stay in".
Jim Allister, leader of the Traditional Unionist Voice (TUV) party, described Mr Cameron's deal as "pitiful".
"Not a single important aspect of our relationship with the EU will change as a result of Mr Cameron's deal," he said.
"Roll on the referendum in which TUV will campaign vigorously, and unitedly, and with others to extricate our nation from the EU. We have nothing to lose but our chains."