Libertas leader Declan Ganley tonight conceded defeat in the North West after losing some 3,000 votes in a recheck.
Mr Ganley confirmed he was bowing out of politics and would not lead a No vote campaign in the next Lisbon Treaty vote.
His defeat paves the way for sitting MEPs Marian Harkin and Jim Higgins of Fine Gael, as well as former MEP Pat "The Cope" Gallagher to take the three seats.
"I will not be involved in the second Lisbon campaign, I've said that upfront. I've got to get back to work," he said.
"I sought a democratic mandate and I didn't get one, and that's how democracy works. And as I said I can take no for an answer."
Mr Ganley was speaking outside the count centre, the TF Royal Theatre in Castlebar, where he was asked if Libertas would continue.
He said: "We'll see, I mean that's something that we'll discuss. There are very many thousands and thousands of people involved in Libertas across the European Union. We'll all be discussing that internally and they'll decide what we do next."
Earlier returning officer Kevin McDermott announced the result of the recheck of the first count of votes in the count centre in Castlebar.
The total valid poll is now 495,307, which is 50 more than the figure announced last night. The extra 50 votes, found in a bundle of 500, belonged to Labour's Susan O' Keefe who was eliminated on the second count.
The new figures did not have an impact on the outcome. Ms Harkin, Mr Higgins and Mr Gallagher are due to be elected by late tonight or the early hours of tomorrow morning.
The rechecking of ballot papers, which began this morning and was scheduled to take two to three hours, continued until just before 5.30pm.
Five Independent candidates have now been eliminated. They are: Michael McNamara (12,744 first preferences), Fiachra O'Luain (6,510), John Francis Higgins (3,030), Noel McCullagh (1,940) and Thomas King (1,124).
The second count is now underway in the TF Theatre.
The count was suspended late last night.
Mr Ganley asked the returning officer, Kieran McDermott, to "check out" a report that "a bin of Ganley votes" had been mistakenly allocated to another candidate, according to Mr Ganley's spokesman.
Mr McDermott announced a re-check of votes last night "in the light of what Mr Ganley has said to us".
But this evening it was announced Mr Ganley's first preference vote dropped by 3,000 to 67,638 after the recheck.
The 3,000 votes belong to the Independent candidate Fiachra O'Luain, who now has 6,510 votes, but has still been eliminated.
On first preferences, Ms Harkin topped the poll with 84,813. Mr Gallagher followed with 82,643 and Mr Higgins with 80,093.
Mr Ganley came fourth.