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The Department of Defense (also called DOD) is a department in the executive branch of the United States Government. It is responsible for military operations and is based out of the Pentagon. The head of the Department of Defense, the Secretary of Defense, is one member of the President's cabinet. The Secretary of Defense in Day 4 and Day 5 (appointed by President Keeler) was James Heller. By Day 6, The Secretary of Defense was Ethan Kanin.

Although the Counter Terrorist Unit is part of the CIA, Secretary Heller took over CTU Los Angeles during Day 4, later transferring to Division. During Day 5, Audrey Raines served as an interagency liaison between DOD and CTU.

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