S-27′s construction was authorized in March 1917, and her keel was laid down on 11 April 1919 by Bethlehem Shipbuilding Corporation's Fore River Shipyard in Quincy, Massachusetts.

Based at New London, Connecticut, through 1924, S-27 transferred to the Pacific in 1925, and, after exercises in the Hawaiian Islands during the spring of 1925, arrived at her new homeport, San Diego, California, in June 1925.

She arrived there on 27 June 1939 and resumed operations off the coast of Southern California, conducting exercises and tests, primarily for the Underwater Sound Training School.

S-27 still was undergoing overhaul when the United States entered World War II with the Japanese attack on Pearl Harbor of 7 December 1941.

Then ordered north to the Aleutian Islands, she departed San Diego on 20 May 1942, proceeded to Port Angeles, Washington, and then continued on to the waters of the Territory of Alaska, where she commenced patrol operations.

On 18 June 1942, she reconnoitered Constantine Harbor, found no signs of enemy activity in the evacuated village there, and moved on to round the southern end of Amchitka, from which she would proceed to Kiska.

S-27 crewmembers reboarded her on 21 and 22 June 1942 to take off more supplies, but thereafter, the presence of poisonous chlorine gas due to the reaction of seawater with her battery cells precluded further visits.

On 24 June 1942, a U.S. Navy PBY Catalina flying boat piloted by Lieutenant, junior grade, Julius A. Raven, USNR, on a routine flight spotted the activity at Constantine Harbor, landed, and took off 15 of the survivors.

The men destroyed all guns they had salvaged from S-27 before they departed, leaving behind nothing except S-27′s abandoned wreck and canned provisions, blankets, and winter clothing.