Paul Robinson and Jermain Defoe are in the squad for the match in Madrid on Wednesday but Ledley misses out this time.
"It's just an old problem where the knee keeps swelling up a bit," explained Ledley after the derby against Arsenal.
"It doesn't give me too much discomfort during games but when we play a lot of games in quick succession, it can be a problem. Other than that it's not too bad."
England boss Sven Goran Eriksson said it was 'a shame' that Ledley was out.
He told the Football Association's website: "In defence, we are obviously missing Sol Campbell. His absence would be felt by any team, and it is shame that we miss Ledley King too.
"But we are lucky to have many excellent players in central defence with Rio Ferdinand, John Terry, Jamie Carragher and Matthew Upson coming back in now too.
"All of them are top players. Wayne Bridge has also come back from injury, which gives us some interesting options on the left side."
England squad:Paul Robinson (Tottenham), David James (Man City), Chris Kirkland (Liverpool), Gary Neville (Man Utd), Phil Neville (Man Utd), Ashley Cole (Arsenal), Jamie Carragher (Liverpool), Wayne Bridge (Chelsea), Rio Ferdinand (Man Utd), John Terry (Chelsea), Matthew Upson (Birmingham), David Beckham (Real Madrid), Frank Lampard (Chelsea), Nicky Butt (Newcastle), Owen Hargreaves (Bayern Munich), Jermaine Jenas (Newcastle), Joe Cole (Chelsea), Shaun Wright-Phillips (Man City), Michael Owen (Real Madrid), Jermain Defoe (Tottenham), Alan Smith (Man Utd), Wayne Rooney (Man Utd).
Ledley King has assured fans that the knee problem keeping him out of England's friendly against Spain this week is not too serious.