Argentine star Lionel Messi was ruled out on Monday by Inter Miami coach Gerardo Martino for the defending champions' Leagues Cup knockout match on Tuesday at reigning MLS champion Columbus.
Messi remains sidelined after suffering a right ankle injury a month ago in Argentina's 1-0 victory over Colombia in the Copa America final.
Messi has not yet trained with his Miami teammates since escaping a walking boot.
The Leagues Cup is a tournament with MLS and Liga MX Mexican clubs, with Messi helping lift Miami to last year's inaugural title.
The showdown match between the Columbus Crew, last year's MLS kings, and Miami, this year's top overall team in the league table, is among eight matches on Monday and Tuesday that will decide quarterfinal pairings on Friday and Saturday.
The round of 16 opens on Monday with the Seattle Sounders playing host to Mexico's Pumas.
Seattle defeated the Pumas in the 2022 CONCACAF Champions League final.
Other knockout matches on Tuesday include Philadelphia at Cincinnati, Cruz Azul against Mazatlan, Tigres against New York City, Toluca at Colorado, Club America at St. Louis and San Jose at Los Angeles FC.