Brazil-born Pepe, who cost Real Madrid 30 million euros from Porto in 2007, damaged knee ligaments in December and has not played since.
Portugal coach Carlos Queiroz produced several surprises with Deportivo Coruna central defender Ze Castro called up even though he did not feature in the squad for any of the qualifiers.
"Of course, it's a surprise as this year I hadn't been called up consistently... I would have had more hopes if the season had gone like I thought it could have, I had plenty of physical problems but now I am at my best," Castro told the SportTV channel.
Queiroz called up four other central defenders, including a surprise selection for Lille's Ricardo Costa.
But like Pepe, Castro can also play in front of the defence as he did in his only cap in a friendly last year.
"When I played for the national side against Estonia, I played as a sweeper, and the coach knows I can play in that position," the 27 year-old Castro added.
Queiroz also included two goalkeepers with little international experience, Porto's Beto and Iraklis' Daniel Fernandes keeping Benfica's Quim and Chelsea's Hilario out.
In midfield, Sporting's Joao Moutinho, who played in five qualifiers but had a low-key season, was the main omission.
His club mate Yannick Djalo was left out of Portugal's frontline, with Queiroz calling up Zenit forward Danny who has returned to form after a knee injury.
The list will be reduced to 23 players by June 1.
Goalkeepers: Eduardo (Braga), Daniel Fernandes (Iraklis), Beto (Porto).
Defenders: Miguel (Valencia), Paulo Ferreira (Chelsea), Ricardo Carvalho (Chelsea), Bruno Alves (Porto), Rolando (Porto), Ricardo Costa (Lille), Duda (Malaga), Fabio Coentrao (Benfica).
Midfielders: Pedro Mendes (Sporting), Pepe (Real Madrid), Ze Castro (Deportivo Coruna), Tiago (Atletico Madrid), Deco (Chelsea), Raul Meireles (Porto), Miguel Veloso (Sporting).
Forwards: Simao Sabrosa (Atletico Madrid), Danny (Zenit St Petersburg), Liedson (Sporting), Hugo Almeida (Werder Bremen), Cristiano Ronaldo (Real Madrid), Nani (Manchester United).
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