Plans to play Villarreal's home game against Atletico Madrid on 6 December in Miami have been abandoned.
A Spanish court has decided not to grant "interim measures" to allow La Liga to stage the game as a decisive case on the issue of matches being held abroad is to take place in early 2020.
The planned game is being blocked by the Spanish Football Association.
The court will make a final ruling on whether the FA can prevent La Liga holding a game in the US in February.
Last year Barcelona abandoned plans to play a game against Girona in Miami because of a "lack of consensus".
Who's who in the argument?
- La Liga - the Spanish League, which represents the top-flight clubs and wants to take a game to the US
- RFEF - the Spanish Football Association, which governs the sport in the country and is currently blocking the US games
- AFE - the Spanish players association, an interested observer
The Spanish Football Association (RFEF) and players' union (AFE) were vocal in their objections to the Girona-Barcelona game.
February's court case is about holding league games abroad and will come down to whether, under Spanish law, the FA has the right to block La Liga - the top league in Spanish football - from holding matches in the US.
On Friday, Madrid's Mercantile Court decided not to issue a temporary ruling allowing the Villareal v Atletico Madrid game to go ahead because it does not want to pre-empt the February case.
The Spanish league said: "La Liga respects this decision, which does not prejudge the substance of the matter, which will be permanently settled in February 2020.
"We hope that from February forth La Liga will be able to organise an official match beyond Spanish borders."
Both Villarreal and Atletico had agreed to the game at NFL side the Miami Dolphins' Hard Rock Stadium, but the RFEF had not granted permission. The game will now be played as scheduled in Villarreal's Estadio de la Ceramica.
La Liga signed up to play one game a season in the US as part of a 15-year deal with media company Relevent in 2018.