Clydesdale Bank Premier League clubs have voted "overwhelmingly" to reject the newco Rangers application.
The SPL announced the news in a brief statement after more than five hours of talks at Hampden today.
SPL chief executive Neil Doncaster earlier hinted the vote could be deferred amid fears over the financial consequences of the Ibrox club starting their new life in the Third Division, but clubs carried through their vow to deny Charles Green's bid.
An SPL statement read: "At today's General Meeting, SPL clubs today voted overwhelmingly to reject the application from Rangers newco to join the SPL."
Clyde had previously stated their objection to voting Rangers into the First Division and they stuck to that stance today.
The Cumbernauld-based club said the consequences of refusing the plan would be a loss of the annual £2million settlement payment from the SPL.
They accused the SFA of "absent leadership" and added: "There will be no winners. Any level of integrity for the sport will be lost by one outcome and financial collapse, we are told, will fall upon the SPL with the other.
"The board of the SFL are being put under intolerable pressure by the other bodies looking to avoid the implications of properly applying their own governance procedures.
"In summary, the SFA implication is that there will be no entry to the SPL.
"The SPL implication is that it therefore has to be SFL1 with a bit of restructure, or an SPL2 with the rest of the SFL cut adrift. There were no other options.
"Whilst Stewart Regan said that the SFA did not favour an SPL2, there was no equivalent abhorrence of that proposal as was attached to the proposal for a newco in the SPL, leaving the implication that the door remains wide open for the SPL to secure their £16million with or without the SFL."
St Mirren confirmed on their website they had voted to reject the application as previously intimated.
Hopes from the SPL clubs that the SFL would back the First Division option suffered a blow during the meeting when Dunfermline and Clyde confirmed their opposition today and Partick Thistle said they could not support the plans, although they called for radical overhaul.
However, Clyde also claimed they had been left in little doubt following an SFL meeting on Tuesday that the Third Division was not a viable option.
The club said: "The SPL implication is that it therefore has to be SFL1 with a bit of restructure, or an SPL2 with the rest of the SFL cut adrift. There were no other options."
The SFL were expected to hold a board meeting tomorrow where a date could be set for a vote on the newco admission.