Portuguse winger Nani was on Tuesday ruled out of this year's FIFA World Cup with a left shoulder injury.
"After tests we conclude he is unfit to participate in the World Cup," the Portuguese Football Federation said in a statement.
Nani picked up the injury in training on Friday in Lisbon before flying to South Africa.
During the flight to Johannesburg, he had been confident he would recover in time.
He will be replaced by Benfica's Ruben Amorim, the Federation added.
Portugal play their first tournament match on June 15 against the Ivory Coast in Group G, which also includes Brazil and North Korea.