She was launched on 2 July 1957, sponsored by Mrs. John A. Moore, widow of the last commanding officer of the previous USS Grayback (SS-208), and commissioned at Mare Island on 7 March 1958.

While operating out of Naval Base Ventura County in Port Hueneme, California, in September 1958 she conducted the first successful launching of a Regulus II missile from a submarine, whose predecessor, Regulus I, had led to the capability of navies to attack land bases since its deployment in 1957.

Departing San Diego, California, on 30 October, Grayback arrived at Pearl Harbor on 8 November for a month of exercises and maneuvers before returning to Mare Island for her "10,000 mile checkup."

On her nine patrols she spent more than 20 months at sea and logged over 130,000 miles (209,000 km) on deterrent missile strike missions.

Main propulsion was lost for a short time, was restored, and Grayback returned to Pearl Harbor under her own power.

[2] As more Polaris missile submarines became operational, they assumed the deterrent functions previously assigned to Grayback and her sister ships.

(The Naval Vessel Registry entry for Grayback shows that at one point she was classified as a "plain" transport submarine, an APSS.

The conversion heightened her sail by ten feet, added two auxiliary tanks to the forward end of the engine room (increasing the length of the boat by 12 feet), and converted the missile chambers to carry 67 embarked troops and SEAL Swimmer Delivery Vehicles (SDVs), including a decompression chamber in the starboard hangar.

In June 1972, the Grayback carried a team of Navy SEALs into the coastal waters of North Vietnam as part of Operation Thunderhead.

In January 1982 five U.S. Navy divers died when a vacuum was inadvertently drawn in a chamber on Grayback off the coast of Subic Bay.