DUP leader Peter Robinson says there are "too many other unionists standing for Europe" for his party to field a second candidate.
He made the statement as he accompanied his party's sitting MEP Diane Dodds to lodge her election papers yesterday.
Mrs Dodds was the last candidate to nominate.
Until last month, there had been speculation that the DUP might field a running mate for her in the forthcoming European poll.
Mr Robinson said: "There was an argument that a second DUP candidate could have returned two DUP MEPs and secured the second seat for unionism.
"However, in an already crowded field, the addition of another unionist candidate would only have increased the chances of a second non-unionist being elected.
"The DUP put unionism first," he added.
"It is a sad reflection on unionism that there could be as many as six unionists on the ballot paper, while republicans and nationalists will unite around Sinn Fein and the SDLP. Shredding the unionist vote will only assist the non-unionist candidates."
Mr Robinson pointed out that Mrs Dodds had been "the most active Northern Ireland MEP, both in the parliament and in Northern Ireland".
This was based on the MEP ranking website, which put her ahead of both fellow MEPs Jim Nicholson and Martina Anderson in terms of parliamentary activity. It also put her in the top 10% of MEPs across Europe.
Mrs Dodds took a swing at Ms Anderson, her Sinn Fein rival, who had promised that her party would "put Ireland first in Europe".
"On May 22 I am seeking support from the people of Northern Ireland and I will put Northern Ireland first within the European Parliament," she said.
"This is in stark contrast to the utter folly of Sinn Fein's candidate.
"The Sinn Fein MEP's decision to put 'Ireland first' costs Northern Ireland."
She later called for police to take action following yesterday's burial of murder victim Tommy Crossan, where masked men flanked the coffin of the former Continuity IRA commander.
"Those who took the life of this individual must be found and imprisoned, but it is also wrong to celebrate the life of a terrorist through such a show of strength," she said.
Her election agent Lord Morrow revealed that several of her election posters had been stolen in the Dungannon and Donaghmore areas.
"While I may not be the biggest fan of election posters, they are part of our electoral process.
"I have reported this incident to the police," he said.
European elections will be held on May 22. Nominations closed at 4pm yesterday. There are a total of six pro-Union candidates – Diane Dodds (DUP); Jim Nicholson (UUP); Tina McKenzie (NI21); Mark Brotherson (Conservatives); Jim Allister (TUV), and Henry Reilly (Ukip). The non-unionist candidates are Anna Lo (Alliance), Alex Attwood (SDLP), Ross Brown (Green) and Martina Anderson (Sinn Fein). Ms Anderson, Mrs Dodds and Mr Nicholson currently hold NI's three seats.