After fitting out at New London, "Tambor" got underway on 6 August 1940 for her shakedown cruise which took her to New York City, Washington, D.C., Morehead City, North Carolina, and Houston, Texas. Following further training off Colón, Panama, the submarine returned to New London, Connecticut, before holding her acceptance trials and undergoing a post-shakedown overhaul at the Portsmouth Navy Yard in Kittery, Maine. After conducting live-fire trials on the effectiveness of depth charges, the first of their kind in the U.S. Navy, "Tambor" reported in May 1941 to the Submarine Force, Pacific Fleet, and the command of Rear Admiral Thomas W. Withers, Jr. (COMSUBPAC).