Players who opt to compete on the breakaway LIV Golf circuit in 2027 will not be allowed to be members of the European Tour, officials said on Monday.
Some of the world's leading players have been given conditional releases in recent seasons, which meant they could compete on both circuits.
But DP World (European) Tour chief executive Guy Kinnings said players must now "make a choice".
In order to be eligible for Europe's Ryder Cup team, players must be members of the European Tour.
"Players need to make a choice," Kinnings told the BBC. "We have rules and regulations which are there to protect our tour and which our members must all abide by.
"Players who are not willing to comply with our rules and regulations should not be seeking to join our tour."
LIV players such as Jon Rahm and Tyrrell Hatton are currently eligible to play on the European Tour under one of its membership categories.
LIV Golf's future is uncertain after it filed for bankruptcy protection in September following the withdrawal of financial backing from Saudi Arabia's Public Investment Fund.
New lead investor BC Partners Credit has pledged an initial funding commitment of a potential $300 million to help LIV relaunch in 2027.
Two-time major winner Rahm is set to quit LIV Golf after refusing to accept new terms.