In this study, we examined hamstring muscle activation at different running speeds to help better understand the functional characteristics of each hamstring muscle. Eight healthy male track and field athletes (20.1 +/- 1.1 years) performed treadmill running at 50%, 75%, 85%, and 95% of their maximum velocity. Lower extremity kinematics of the hip and knee joint were calculated. The surface electromyographic activities of the biceps femoris and semitendinosus muscles were also recorded. Increasing the running speed from 85% to 95% significantly increased the activation of the hamstring muscles during the late swing phase, while lower extremity kinematics did not change significantly. During the middle swing phase, the activity of the semitendinosus muscle was significantly greater than that of the biceps femoris muscle at 75%, 85%, and 95% of running speed. Statistically significant differences in peak activation time were observed between the biceps femoris and semitendinosus during 95%max running (P < 0.05 for stance phase, P < 0.01 for late swing phase). Significant differences in the activation patterns between the biceps femoris and semitendinosus muscles were observed as running speed was increased, indicating that complex neuromuscular coordination patterns occurred during the running cycle at near maximum sprinting speeds.