The 17-year-old winger, reportedly a target for Manchester United and Arsenal during the transfer window, was one of three new faces named by Stuart Pearce for the game in Empoli.
Chamberlain was joined by Burnley striker Jay Rodriguez and Middlesbrough defender Joe Bennett in a 22-man party for what is England's first match of 2011.
As expected, Jack Wilshere was not selected for the match, with Fabio Capello having indicated the Arsenal midfielder will start the full international with Denmark the following evening.
But Sunderland's Jordan Henderson returns to the Under-21s following a disappointing senior debut against France in November, while full-backs Kieran Gibbs and Micah Richards and goalkeeper Scott Loach also dropped back down.
Daniel Sturridge, who joined Bolton on loan from Chelsea on Monday, was handed a recall as was West Ham's James Tomkins - his first for more than a year - and Aston Villa's Fabian Delph.
But Sturridge's Stamford Bridge team-mate Josh McEachran, who earned his maiden call-up for November's friendly with Germany, was omitted.
As well as Wilshere, Pearce will be without the injured Danny Welbeck, Danny Rose, Michael Mancienne, Tom Cleverley and Phil Jones.
He told www.thefa.com: "We're missing a number of players through injury, but it will give me a chance to look at a few new players. Rodriguez has been playing well for Burnley and deserves an opportunity with the under-21s.
"Likewise, Joe Bennett at Middlesbrough has previously played for the under-19s and his performances for Tony Mowbray have been impressive. I also saw Southampton play Manchester United in The FA Cup on Saturday and Alex Chamberlain is showing a lot of promise so we thought we'd bring him into the squad too."
Source: PA
Source: PA