Three Fianna Fáil Senators - Jim Walsh, John Hanafin and Labhrás Ó Murchú - have resigned the party whip over their opposition to the Civil Partnership Bill.
In a letter addressed to Seanad Chief Whip Senator Diarmuid Wilson, the Senators said the move was "a matter of regret".
"We write to advise you that we feel it incumbent upon us to relinquish the Whip with effect from the commencement of the Order of Business today, as we have tabled amendments to the Civil Partnership Bill," the Senators wrote.
"It is a matter of regret that we conscientiously find this to be the only appropriate avenue open to us, and, we would like to offer our apologies to both the Taoiseach and the Minister for Justice for any difficulties that this might present."
The Senators said that they would continue "to support the continuing efforts of the Government to restore a functional banking system, improve the economy, and fulfil the Programme for Government".
Earlier in the Seanad, Senator Walsh said while he can accept diversity in society, he cannot accept anything that damages the institution of marriage.
"Existing marriage rights should remain unique to marriage, because of the uniquely pro-child aspects of marriage," Senator Walsh said.
"It is not discrimination to treat a unique institution - like the marriage of a man and a woman - in a unique manner."
Senator Walsh's comments were made as a proposal to allow a free vote on the Civil Partnership Bill in the Seanad was been defeated by 44 votes to six.
The legislation gives legal rights to gay couples for the first time.
Senator Walsh said he is compelled to follow his conscience - and he is appealing for respect for his decision.