Colombia and Ecuador have both made last-minute replacements to their World Cup squads because of injuries players suffered in warm-up matches.
Segundo Castillo, a veteran midfielder who played for Ecuador at the 2006 World Cup in Germany, has failed to recover from a knee injury he suffered on 31 May.
Castillo sustained the injury in a tackle with Mexico's Luis Montes during a friendly in Arlington, Texas.
Montes broke his leg and was immediately withdrawn from Mexico's squad after their 3-1 win.
Coach Reinaldo Rueda called up Chivas USA midfielder Oswaldo Minda in Castillo's place for the tournament starting on Thursday.
Ecuador, due to travel to Brazil on Tuesday, are drawn in Group E with Switzerland, Honduras and France.
Colombia midfielder Aldo Leao Ramirez took a knock during training last week, missing their 3-0 win in a friendly over Jordan in Buenos Aires on Friday.
He is being replaced by winger Carlos Carbonero, who helped River Plate win the Argentine league title last month.
Ramirez is the fourth player Colombia have lost to injuries, after top striker Radamel Falcao, midfielder Edwin Valencia and defender Luis Perea.
Colombia face Greece, Ivory Coast and Japan in Group C.