In the early days of World War II, prior to the U.S. becoming a combatant, DC-3s and some DC-2s were used to survey "airway routes" that later military transports would use to move troops and materials.

On June 26, 1941, Pan American Airways contracted with the U.S. government to establish a ferry service and air transport service from Miami to the Middle East, the west coast of Africa and on to Khartoum. Pan American Ferries, Inc. handled delivery of many of the aircraft supplied to Britain in Africa. The first DC-3s service from Bathurst, in Gambia, to Khartoum, started on October 21, 1941, and there were ten Douglas transports on the line by December 7, 1941.

Pan American Airways began flights from Miami, Florida, and Natal, Brazil, to connect with service across the South Atlantic, using five C-53s in February 1942. They later expanded the number of aircraft on that route to fourteen.

Eastern Airlines also began a ferry service from Miami to South America, Trinidad, Puerto Rico, and Nassau, under contract with the Air Service Command on May 1, 1942, using DC-3s.

Northeast Airlines' crews flew a C-39 and C-53 along the Eastern seaboard to Newfoundland and Labrador, charting routes to Europe. The aircraft would land at such places as Prestwick, England, Trinidad, British Guiana, Gander, Goose Bay, Ascension Island and dozens of other far-away places where they established radio beacons to guide the thousands of flights that were to follow weeks later. These flights would fly priority cargos, or VIPs and a few made secret wartime missions. In June 1942, Northeast Airlines received a contract to fly the Goose Bay,

Greenland, Iceland and Prestwick, England route using C-53s

Because of the proximity of Japan to Alaska, and the Aleutian Islands, Northwest Airlines received a survey contract to map routes to Fairbanks, Alaska. By September 1942, they were flying 15 DC-3s from Great Falls, Montana to Fairbanks. Western Airlines flew four routes from Ogden, Utah to Anchorage. American Airlines flew to Iceland, Western Airlines to Edmonton, Canada and United Airlines flew from Dayton, Ohio to Alaska, all using C-53s.

These air routes were of invaluable assistance when on June 3, 1942, the U.S. rushed troops on DC-3s to the Aleutian Islands to counter an attack on Dutch Harbor. The attack was short-lived and prevented a threatened occupation of the islands which may have altered the course of the war.

Of the 322 commercial airplanes in the United States, in 1941, at least 289 of them were DC-3s/DSTs. Since Douglas had not filled all the Army Air Corps orders for transport aircraft, the War Department appropriated, 94 DC-3 transports (leaving the airlines with less than 200 to carry on domestic service).

Many of the drafted DC-3s were leased back to the airlines. Some flew military transport routes in the military markings of the Air Transport Command (ATC), and others flew commercial routes assigned by the government in civilian markings.

These drafted DC-3s were designated variously C-48s to C-53s. At this point, the DC-3 had already flown 300,000,000 miles, and had more than 2,000,000 hours in the air.

The DC-3 had been flying from December 1935, to December 1941, a full 72 months, and was a well-tested machine. In that period, the few bugs in it had been shaken loose.

The oldest route under the ATC command was Pan American's Africa Service that stretched from Bathurst, Gambia to Cairo, Egypt. Pan American operated eleven DC-3s along this route and kept the civilian markings on the aircraft. In a typical example of government war-time communications, the War Department ordered Pan American's trans-African service to become a military operation as soon as possible. The DC-3s were allocated USAAF serial numbers on March 14, 1942, and when the Army went looking for the airplanes they realized that the eleven DC-3s had been transferred to the RAF ten days earlier. The USAAF then rolled in eleven C-53s and assigned them to the route.

The Air Transport Command racked up some impressive statistics. One airline, Western Airlines (later to merge with Delta) achieved the highest aircraft usage in the ATC. By the end of the war, Western Airlines had carried 22 million tons of goods, and flew 67 million passenger-miles without a hitch. Their DC-3s logged on the average, 15-19 hours a day in the air.

The DC-3 converted to a military transport (C-47, 53 etc.) was one of the four pieces of equipment that General Eisenhower said had one the war. The others were the bulldozer, jeep, and 2 1/2 ton truck.