Chelsea duo Marcos Alonso and Alvaro Morata have been included in Luis Enrique's first Spain squad, with only two Barcelona players, Sergi Roberto and Sergio Busquets, in the 24-man selection.
Morata, left out of Julen Lopetegui's squad for Russia, is among 10 players to make a return for the Nations League game against England at Wembley on Sept. 8 and the home match with Croatia three days later.
"These two games against World Cup semifinalists England and Croatia, are going to be very important to see where we are and how hungry we are," Luis Enrique said at a news conference.
"I've dedicated a lot of hours and days to execute this squad. I'm eager to see all the players and set up the path we have to follow. It's a balanced squad, with a balanced age of 26.
"Morata is a player that offers a lot of possibilities. He didn't finish the season in the best way, and that is why he missed the World Cup.
"I've seen improvement at the start of this season, and I hope he will continue in this ascending line."
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Morata is one of four Chelsea players in the 24-man squad, while Real Betis goalkeeper Paul Lopez, Valencia defender Jose Luis Gaya and Real Madrid midfielder Dani Ceballos have received their first call-ups.
Jordi Alba, Koke and Iago Aspas, regulars under Lopetegui, have been dropped from the squad.
"It's a very special day for those that are on the list and I have nothing to say regarding those who are not," Luis Enrique said. "I haven't spoken to any player, that is my style. I didn't think it was necessary to speak to any player."
He said Real Madrid winger Isco remained a key part of his thinking, adding: "I see Isco as a very complete player. He is one of the most crucial players in the national team and in Spanish football."
Luis Enrique refused to confirm whether Manchester United goalkeeper David De Gea, criticised for mistakes at the World Cup, would be Spain's No. 1.
"I have three goalkeepers," he said. "I like the three I've chosen and others could have also been included. You will have to wait and see who will be the No. 1."
Meanwhile, Napoli defender Raul Albiol returns, having not featured for La Roja since the 2014 World Cup.