The Teachers Union of Ireland has rejected the new public service pay agreement by a margin of 87% to 13%.
The Irish National Teachers Organisation has already rejected the deal.
If the ASTI reject it in their ballot in October, all three unions will be opposing the Government's pay proposals.
However, it remains to be seen whether they will contemplate industrial action in pursuit of their grievances, the main one being the lower pay rates for more recent recruits.
In a separate TUI ballot, the 18,000 members of the union voted in favour of industrial action by a margin of 81% to 19%.
Despite the TUI'S rejection, the successor to the Lansdowne Road public service pay agreement is now certain to be ratified by public service unions this Monday.
In the ratification process, each public service union is given a weighted number of votes out of a total of 2892. In order to be passed, the deal would need at least 1447 votes.
Given that the two largest unions SIPTU (719 votes) and IMPACT (606 votes) had already balloted in favour of the deal, it was already well on the way to formal approval.
Today, the Public Service Executive Union (113 votes) backed the deal, with 85% in favour - bringing the number of votes in favour to 1544 so far - guaranteeing ratification.
Also voting yes so far are the Prison Officers Association (34 votes), the Association of Higher Civil and Public Servants (32 votes), the Medical Laboratory Scientists Association (19 votes), the Technical Engineering and Electrical Union (10 votes), the Building and Allied Trades Union (5 votes), the Veterinary Officers Association (3) and the plasterers' union OPATSI (3 votes).
Only three unions have voted no so far: the TUI (158 votes), INTO (314 votes) and Unite (60 votes).
Tomorrow the Irish Nurses and Midwives Organisation (390 votes), the Civil Public and Services Union (128 votes) the Irish Medical Organisation (68 votes) and the Irish Federation of University Teachers (20 votes) will reveal the outcome of their ballots.
The Association of Secondary Teachers in Ireland will not complete a ballot until next month, and is recommending rejection.
Assuming the deal is carried, unions who reject it will then have to decide whether to abide by the aggregate vote of their majority union colleagues.
If they do not, they will not be entitled to avail of the benefits of the new agreement - including further pay restoration.
It will also remain to be seen if they will contemplate industrial action in pursuit of their grievances.
Three other groups of public servants outside the Irish Congress of Trade Unions are also considering the LRA2 proposals.
The Garda Representative Association today decided to recommend acceptance.
The Association of Garda Sergeants and Inspectors will reveal its position on the deal at an Autumn Seminar in Athlone on 16 October.
The Psychiatric Nurses Association has not yet decided when to ballot on the deal.
The proposals were negotiated between the government and public service unions earlier this summer.
They set out a pathway for further restoration of pay cuts imposed during the economic crisis, as well as provisions on pensions and work practices.
However, they do not offer an immediate solution to the problem of lower pay rates for public servants recruited after 2012 - and will retain the existing pension levy for workers with fast accrual pension arrangements, who can retire earlier with full pension entitlements.