In October 1939, No. 264 (Madras Presidency) Squadron was reformed at RAF Sutton Bridge to operate the Defiant. Initial training and development of tactics began with other aircraft as it received its first Defiants only in early December at Martlesham Heath. They began night fighter training in February 1940. The squadron tested its tactics against British medium bombers – Hampdens and Blenheims – and 264's CO flew against Robert Stanford Tuck in a Spitfire, showing that the Defiant could defend itself by circling and keeping its speed up. By March, 264 Squadron had two flights operational with Defiants and No. 141 Squadron received its first Defiant. When the Defiant was first introduced to the public, the RAF put out a disinformation campaign, stating that the Defiant had 21 guns: four in the turret, fourteen in the wings and three cannon in the nose.
The first operational sortie came on 12 May 1940. Defiants flew with six Spitfires of 66 Sqn, and a Ju 88 was shot down over the Netherlands. The following day, in a patrol that was a repetition of the first, Defiants claimed four Ju 87s, but were subsequently attacked by Bf 109Es. The escorting Spitfires were unable to prevent five of the six Defiants being shot down by a frontal attack.
During the evacuation of the British Expeditionary Force from Dunkirk, the squadron was forward-based at RAF Manston, as one of the 16 squadrons that No. 11 Group had available to cover the evacuation. On the 27th 264 Sqn claimed three He 111 and two damaged. On the 28th, shortly after takeoff, ten Defiants were attacked by about 30 Bf 109s – forming a circle, they claimed six German fighters for the loss of three Defiants.
The Defiant was initially successful against enemy aircraft. Its best day was 29 May 1940, when No. 264 Sqn claimed 37 kills in two sorties: 19 Junkers Ju 87 Stuka dive bombers, mostly picked off as they came out of their dives, nine Messerschmitt Bf 110 twin-engined heavy fighters, eight Bf 109s, and a Ju-88. One Defiant gunner was lost after he bailed out, although the aircraft made it back to its base to be repaired.