It was returned to duty in 2007 as a replacement of the Alabama ANG 160th Fighter Squadron so the state could honor the legacy of the Tuskegee Airmen.

The Group was transferred to Selfridge Army Air Base, near Detroit, Michigan in March 1943 after the decision was reached to deploy the unit.

However, the unit was delayed again going into combat, and also was sent back to Selfridge upon completion of training at Oscoda AAF to perform Air Defense flights over the Detroit area beginning in July 1943.

For six months, the unit remained at Selfridge until pressure on the Army led to the decision to deploy the 100th to the Fifteenth Air Force in Italy at the end of 1943, under the command of Robert B. Tresville, to support the strategic aerial bombardment campaign being carried out by Boeing B-17 Flying Fortress and Consolidated B-24 Liberator Heavy bombers from newly established air bases in the Foggia, Italy area.

At Lockbourne, the Wing was equipped with new F-47N Thunderbolts that were designed for very-long range flights in the Pacific Theater to attack ground targets in the Japanese Home Islands.

In 1954, the squadron moved to Pease AFB, New Hampshire; received Boeing KC-97 Stratofreighters and performed aerial refueling with SAC's Boeing B-47 Stratojet units, deploying frequently to England and Morocco in North Africa to operate from what were called "REFLEX" bases to refuel SAC bombers prior to their planned flights into Communist-controlled Eastern Europe and the Soviet Union.

The 100th Air Refueling Squadron continued to operate from Pease until the end of the B-47 and REFLEX era in 1966, at which point the unit was inactivated.

Consisting of Boeing KC-135 Stratotankers from the 100th Air Refueling Wing, RAF Mildenhall, England, the squadron was active between April–June 1999 when the Kosovo Campaign ended.

After the Kosovo Campaign, the 100th was again re-activated as a flying training squadron at Randolph AFB, Texas, equipped with Beechcraft T-6 Texan II, Raytheon T-1 Jayhawk and Northrop T-38C Talon trainers.

From the onset the squadron started training on the block 30 version of the General Dynamics F-16C/D Fighting Falcon that carried over from the 160th FS.

The squadron deployed to Romania in August 2012 to participate in Dacian Viper 2012, a three-week joint exercise with the Romanian Air Force.