Launched as J. Howland Gardner, the third Jamestown (AG-166) ship was actually a converted Liberty ship. It was launced under the Maritame Commission contract by the New England Shipbuilding Corporation in South Portland, Maine on July 10, 1945. The ship was sponsored by Mrs. George W. Elkins of Newport, Rhode Island.

This liberty ship was completed on August 14 and was then chartered under general agency agreement by the Waterman Steamship Co. Then on June 17, 1945 it went into the Maritime Reserve Fleet, where it was chartered by U.S. Navigation Co., on February 3, 1947, and then by the South Atlantic Steamship Lines on October 20, 1948.

J. Howland Gardner returned to the Maritime Reserve Fleet at Beaumont, Texas, where, on August 10, 1962, she was acquired by the Navy. Upon this acquisition, the Navy renamed the ship to Jamestown and designated it AG-166 on March 6, 1963. On December 13 of the same year, the ship was commissioned at Norfolk Navy Yard and Commander Alan J. Kaplan was put in command. Norfolk remained her home port when this ship was assigned to Service Squadron 8, Service Force, Atlantic Fleet. Jamestown's mission was "to conduct technical research operations in support of U.S. Navy electronic research projects, which include electromagnetic propagation studies and advanced communications systems such as satellite communications."

On January 20, 1964, the USS Jamestown headed off for Guantanamo Bay, Cuba where she underwent two weeks of intensive shakedown training. When Fidel Castro had shut off all fresh water to her base, the ship was there and was ready to evacuate all American families from there. After undergoing the training, Jamestown had visited Kingston, Jamaica and Key West in South Florida. On February 27, she returned to her home port in Norfolk.

On April 1, Jamestown was redesignated AGTR-3, and eight days after which, she departed on her very first deployment. She visited Gibraltar, Valleta, Malta, Aden, Capteown, and Freetown, Sierra Leone. Some months after, on August 17, she again returned to Norfolk. For some weeks, she began preparation for heading off to the African coast. On November 2, she finally left Norfolk for Dakar, Senagal and Capetown, South Africa. There she was serviced and learned about electronic communications.

On February 6, 1965, the Jamestown returned to Norfolk, and spent her spring in the Caribbean. She then went through the Panama Canal and went on a cruise along the western coast of South America and reached Valparaiso, Chile on the ninth of June. About a month and a half later, on July 23, she returned to Norfolk. Three months passed, and the Jamestown ventured off for the Far East reaching the Subic Bay in the Philippines on December 29. Operating in the South China Sea, the Jamestown gathered valuable information for the Navy's ships which were engaged in the Vietnam War. Through mid-1967, she stayed in this area, often operating in the war zone.