Studies of skeletal muscle from rodents performed both in vivo and in vitro suggest a regulatory role of glycogen synthase kinase (GSK) 3 in glycogen synthase (GS) activation in response to insulin. Recently, hyperinsulinemic clamp studies in humans support such a role under nearly physiological conditions. In addition, in rats the activation of GS in skeletal muscle during treadmill running is time-related to the deactivation of GSK3. We investigated whether GSK3 was deactivated in human muscle during low- (approximately 50% VO2max for 1.5 h) and high-intensity (approximately 75% VO2max for 1 h) bicycle exercise as well as food intake. We observed a small but significant increase in GSK3alpha (10-20%) activity in biopsies obtained from vastus lateralis after both low- and high-intensity exercise, whereas GSK3beta activity was unaffected. Subsequent food intake increased Aktphosphorylation (approximately 2-fold) and deactivated GSK3alpha (approximately 40%), whereas GSK3beta activity was unchanged. GS activity increased in response to both exercise and food intake. We conclude that GSK3alpha but not GSK3beta may have a role in the regulation of GS activity in response to meal-associated hyperinsulinemia in humans. However, in contrast to findings in muscle from rats, exercise does not deactivate GSK3 in humans, suggesting a GSK3-independent mechanism in the regulation of GS activity in muscle during physical activity.