Using the perfused rat hindlimb preparation, the role of insulin in the regulation of glycogen metabolism in voluntary skeletal muscle has been characterized. A maximally effective concentration of insulin (1 mU/ml) caused a threefold increase in glucose clearance by 5 min. However, the -glucose-6-P/+glucose-6-P activity ratio of glycogen synthase was not significantly increased before 20 min. Insulin concentrations as low as 0.1 mU/ml significantly modified the glycogen synthase activity ratio and the half-maximal activation constant (A0.5) for glucose-6-P at 30 min, but had no effect on tissue cAMP. These changes were not dependent on the presence of glucose and were not modified by fasting. These results indicate that high physiological concentrations of insulin activate glycogen synthase in voluntary skeletal muscle and that this effect is independent of changes in glucose uptake or tissue cyclic AMP.