Mr. Scott has said that he wanted no part of the project, calling it a “boondoggle,” because he believed Florida would be responsible for cost overruns during construction and subsequent subsidies that might be necessary to keep it operating if the link was unable to pay for itself in terms of ridership.

Mr. Scott’s decision has been opposed by both Democratic and some Republican lawmakers, who have said the high-speed link would give the state’s staggering economy a boost.

And in the last several weeks, in an effort to allay Mr. Scott’s concerns, the federal Department of Transportation, along with the mayors of Tampa and Orlando and other Florida political leaders, had crafted an arrangement in which the contractor hired to build the rail line would have been responsible for all of its construction and operating expenses as a way to further insulate taxpayers if something went wrong.

It was not immediately clear why Mr. Scott rejected the proposal.

After Mr. Scott’s original rejection of the rail project on Feb. 16, two Florida state senators  Arthenia Joyner, a Democrat and Thad Altman a Republican  filed a lawsuit against the governor, maintaining that he had exceeded his constitutional authority and had violated the separation of powers under the state’s Constitution.

On Friday, the Supreme Court said those claims had not been supported.

The 85-mile Tampa-to-Orlando route, on which trains would have traveled as fast as 170 miles per hour, was one of two high-speed lines approved by Congress. The other would connect San Francisco and Los Angeles at speeds up to 225 miles per hour. That project has received federal pledges of about $3 billion, though the cost has been estimated to be at least $43 billion.