Berkeley began playing roles in 1981, with early appearances in M*A*S*H, Cagney & Lacey, Remington Steele, Miami Vice, Moonlighting and The A-Team. Although not becoming a household name, Berkeley's face was increasingly recognizable into the 1990s. His later television guest roles included The X-Files, CSI, ER, 24 and Law & Order.
On the big screen, Berkeley has appeared in North Country, Terminator 2: Judgment Day, Mommie Dearest, Phoenix, Kick-Ass, A Few Good Men, The Rookie, Candyman, Apollo 13, Leaving Las Vegas, Gattaca, The Rock (uncredited), Air Force One, Sid and Nancy, Spawn, Amistad, Shanghai Noon, Barb Wire and Timecode. Berkeley has the distinction of appearing in both the made-for-television film L.A. Takedown in 1989 and its 1995 critically acclaimed theatrical remake Heat, both directed by Michael Mann. Several of his earlier roles were in films by director Alex Cox.
In 2001, Berkeley became a recurring guest star and later on as a series regular on 24 in the role of George Mason, the head of the Counter Terrorist Unit.
Berkeley has provided voices for animated series such as Aaahh!!! Real Monsters, Teen Titans and Gargoyles. He voiced Quentin Beck/Mysterio in The Spectacular Spider-Man and Captain Atom in the animated movie Superman/Batman: Public Enemies.
He portrayed the mysterious John Smith on the CBS drama Jericho.
In 2010, he received one of his best known roles - that of Percy Rose in the CW action-thriller series Nikita. He portrayed the character as a series regular in the show's first two seasons as well as being the series' de facto main antagonist of its first two seasons.
In 2013, Berkeley won the Streamy Award for "Best Male Performance, Drama" his starring role in the acclaimed webseries, The Booth at the End.