Palacio, who scored twice for his club side Genoa in a 3-0 win over Catania on Wednesday, last played for Argentina against Brazil in June 2008.
Zanetti and Cambiasso's absence reflects the dismal start to the season made by their Serie A club with four defeats and a draw in five competitive matches.
Manchester City striker Carlos Tevez was also left out as coach Alejandro Sabella named the squad for his first competitive matches in charge since replacing Sergio Batista after the Copa America.
Argentina host Chile on October 7 and visit Venezuela four days later as they kick off their marathon campaign to qualify for Brazil in 2014.
Nine South American teams play in a single group while Brazil qualify automatically as hosts.
Sabella is due to name a separate squad of home-based players on Sunday for next Wednesday's friendly away to Brazil.
Goalkeepers: Sergio Romero (Sampdoria), Mariano Andujar (Catania)
Defenders: Pablo Zabaleta (Manchester City), Nicolas Burdisso (AS Roma), Federico Fernandez (Napoli), Martin Demichelis (Malaga), Nicolas Otamendi (Porto), Nicolas Pareja, (Spartak Moscow), Marcos Rojo (Spartak Moscow), Emiliano Insua (Sporting)
Midfielders: Javier Mascherano (Barcelona), Ever Banega (Valencia), Fabian Rinaudo (Sporting), Ricardo Alvarez (Inter Milan), Jonas Gutierrez (Newcastle United), Jose Sosa (Metalist Kharkiv), Javier Pastore (Paris St Germain), Angel Di Maria (Real Madrid)
Forwards: Nicolas Gaitan (Benfica), Lionel Messi (Barcelona), Gonzalo Higuain (Real Madrid), Sergio Aguero (Manchester City), Rodrigo Palacio (Génoa), Eduardo Salvio (Atletico Madrid)
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