The PGA Tour has suspended all of its members who are playing in this week's Saudi-funded LIV Golf Invitational.
The tournament, being held at Centurion Club near London, is the most lucrative in the history of the game with a $25m (£20m) prize fund.
Six-time major winner Phil Mickelson is among the biggest names to be affected.
LIV Golf issued an immediate reply calling the PGA Tour "vindictive" and said this "deepens the divide between the Tour and its members".
LIV Golf, which is backed by Saudi Arabia, added: "It's troubling that the Tour, an organisation dedicated to creating opportunities for golfers to play the game, is the entity blocking golfers from playing.
"This certainly is not the last word on this topic. The era of free agency is beginning as we are proud to have a full field of players joining us in London, and beyond."
The PGA Tour released its statement 30 minutes after play had begun at Centurion.
Its decision affects 17 players, including lifetime member Mickelson, although his fellow American Dustin Johnson announced on Tuesday that he had already resigned from the Tour.
The PGA said: "In accordance with the PGA Tour's tournament regulations, the players competing this week without releases are suspended or otherwise no longer eligible to participate in PGA Tour tournament play, including the Presidents Cup.
"The same fate holds true for any other players who participate in future Saudi Golf League events in violation of our regulations."
Crucially, the PGA Tour has closed a loophole that would potentially have allowed players, as non-members, to play in events via sponsor's exemptions.
"These players have made their choice for their own financial-based reasons," said PGA Tour commissioner Jay Monahan in the statement that was issued to all members of the tour.
"But they can't demand the same PGA Tour membership benefits, considerations, opportunities and platform as you. That expectation disrespects you, our fans and our partners."
Sergio Garcia, Martin Kaymer, Graeme McDowell and Lee Westwood had already all resigned from the PGA Tour, but this decision will affect Ian Poulter.
The players will be removed from the FedEx Cup points list following the end of the Canadian Open on Sunday.
The European-based DP World Tour, when contacted by BBC Sport, declined to comment.
