Effects of temperature and pH on the catalytic properties of hexokinase (HK, EC 2.7.1.1) from the flight muscles of Dipetalogaster maximus (Uhler) were studied. The enzyme showed a hyperbolic behavior with its two substrates (glucose and ATP). There was no inhibition by glucose. Apparent Km and Vmax increased as pH increased from 7.0 to 8.5. Catalytic efficiency was lowest at pH 7.0. Km, Vmax, and catalytic efficiency were higher at 37°C than at 30 and 20°C. There was marked inhibition by ATP, which was dependent on pH and temperature. Km values for ATP were reduced and catalytic efficiency increased as pH increased. Lowest Vmax was observed at pH 7.0. At this pH there was 87.3% inhibition by ATP, whereas it was only 5.7% at pH 8.5 (at 30°C). Km, Vmax, and catalytic efficiency were higher at 37°C than at 30 and 20°C. The strong inhibition by ATP detected at 20°C (pH 7.6) almost disappeared at 37°C. Therefore, temperature could regulate hexokinase activity by modulating the inhibition produced by ATP. Glucose utilization and ATP production would be promoted when temperature rises from 30 to 37°C. Because insect thoracic muscles increase their temperature over 30°C during flight, this phenomenon elucidates a mechanism enhancing energy supply for muscle activity.