Her keel was laid down on 16 January 1939 by the Electric Boat Company in Groton, Connecticut. She was launched on 20 December 1939 sponsored by Miss Lucia Ellis, and commissioned on 3 June 1940 with Lieutenant Commander John M. Murphy, Jr. (Class of 1925), in command.
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).
Tambor began a routine peacetime patrol in late November 1941 and was off Wake Island when hostilities with Japan broke out. However, she was forced to return to Pearl Harbor with one engine out of commission. Routed back to Mare Island, where the damage was repaired, the submarine returned to Pearl Harbor in March 1942.
Tambor began her first war patrol on 15 March 1942 when she stood out of Pearl Harbor to reconnoiter the areas around Wake Island, Truk, New Ireland, New Britain, and Rabaul. In all, she made nine attacks; on 16 April, she fired two torpedoes at tanker Kitami Maru. One hit, and she was credited with a sinking; this was not verified by postwar examination of Japanese records. Tambor returned to Pearl Harbor on 12 May, where her skipper criticized the torpedoes.
After refitting, she was then assigned to Task Group 7.1. The group of six submarines sailed for Midway Island on 21 May 1942 to begin patrolling a 150 miles (240 km) circle in anticipation of the invasion fleet intelligence had reported was en route there. At 07:15 on 4 June 90 minutes after first reported contact, COMSUBPAC, Admiral Robert H. English, informed his submarines, waiting until after 11:00 to order them to close. Running surfaced, Tambor was strafed by aircraft