The DP World Tour will not allow any player who signs up to LIV Golf next season to be a member of its circuit in 2027.
The European tour says players must "make a choice" regarding where they play and has taken a significantly tougher stance than it has done in recent seasons.
Players must be members of the DP World Tour to be able to play for Europe in the Ryder Cup.
Deadlines for both tours are in the next five weeks, although there remains uncertainty surrounding LIV Golf's future, despite it securing a possible $300m in funding as it attempts to restructure.
"Players need to make a choice," Guy Kinnings, CEO of the DP World Tour, told BBC Sport.
"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."
In previous years, conditional releases were struck with some top players - including Jon Rahm and Tyrrell Hatton - enabling them to play on both the DP World Tour and LIV.
Next year, however, players will be asked to disclose which tours they intend to play on, and their answer will be taken into account when deciding on their membership.
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Around 15 LIV players, including Rahm, Hatton, Victor Perez and Tom McKibbin, have "status" with the DP World Tour, meaning they have eligibility under one of the membership categories.
Other LIV players, like Joaquin Niemann and Thomas Pieters, do not have such status so would either need to go through qualifying school or sign agreements with the DP World Tour whereby they would receive invitations to a set number of tournaments.
However, those agreements would only be available to players who were not part of LIV in the future.
BBC Sport has been told that the DP World Tour will not accept membership from anyone intending to play on what is being called LIV 2.0.
If players do not disclose their intentions to play LIV and go on to do so, that will be considered grounds on which to have their membership terminated.
LIV Golf filed for Chapter 11 bankruptcy protection in September as it attempted to restructure, following the withdrawal of its multi-billion dollar funding from Saudi Arabia's Public Investment Fund.
Last week, it announced it had secured a possible $300m in financing from BC Partners Credit in order to try to move forward with the 2027 season.
LIV equity offer changes DP World Tour's approach
While the DP World Tour agreed to grant conditional releases with nine players this season, it is understood the change for 2027 is predominantly down to LIV offering players equity in future and a feeling this is incompatible with the European tour being a members organisation.
However, it also comes at a time when the DP World Tour's strategic alliance with the PGA Tour is being negotiated, so the move likely reflects a desire to put itself in the best position possible in those discussions.
Players will have until 16 November to sign up to the DP World Tour for next year.
Under the terms of its court-supervised restructuring process, LIV Golf needs to sign players up by 25 October.
Ted Goldthorpe, head of BC Partners Credit, told BBC Sport last week he was "confident" LIV would have a "critical mass" of players.
But since then two-time major winner Rahm has said he will not continue playing on the LIV tour.
Lawyers representing a number of other players, including fellow major winners Bryson DeChambeau and Cameron Smith, sought clarification from the court that rejection of contracts would equate to "termination", enabling players to hold discussions with third parties about their futures.
Richard Bland is currently the only player to have stated publicly a desire to play on LIV 2.0.
However, Perez indicated last month he hoped there would be a future for the league.