Golfers will be barred from playing on the DP World Tour next season if they choose to join LIV Golf in 2027.
Some of the world's top players were given conditional releases by the European tour in previous seasons, allowing them to compete on both circuits.
But DP World Tour chief executive officer Guy Kinnings has said players must now "make a choice".
In order to be eligible for Europe's Ryder Cup team, players must be members of the DP World Tour.
Kinnings told BBC Sport: "Players need to make a choice. 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's future is in doubt after Saudi Arabia's Public Investment Fund withdrew support at the end of the season and is going through Chapter 11 bankruptcy proceedings in the United States.
The breakaway series has secured an initial funding commitment for a potential $US300 million ($A429 million) investment from BC Partners Credit to help relaunch LIV 2.0 in 2027.
Deadlines for both tours fall next month and players will be asked to disclose which they intend to play on.
LIV players such as Jon Rahm, Tyrell Hatton and Victor Perez and Tom McKibbin are currently eligible to play on the DP World Tour under one of its membership categories.
Rahm is set to quit LIV Golf after refusing to accept new terms offered for its proposed relaunch and is reported to be owed up to $US150m ($A215m) for the remainder of the contract he signed almost three years ago.