As judged by its ability to stimulate glucose uptake and alpha-aminoisobutyric acid (AIB) transport, the sensitivity and the responsiveness of perfused rat muscle to insulin are enhanced after moderately intense treadmill exercise. In fed rats, these enhanced effects of insulin are predominantly restricted to muscles that performed work as evidenced by glycogen depletion. The present study was designed to examine the relationship between glycogen depletion per se and the postexercise changes in insulin action. Toward this end, fed and 48-h starved rats were run on a treadmill for 45 min at moderate intensity, and glucose and AIB uptake were then assessed using the isolated perfused hindquarter preparation. Glycogen is depleted in red muscles such as the soleus and red fibers of the gastrocnemius in fed rats immediately after such exercise, whereas, in starved rats, muscle glycogen is unchanged. As previously shown, the stimulation by insulin of glucose utilization by the hindquarter and AIB transport into red muscles was substantially increased in fed rats after the treadmill run. This was due to increases in both insulin sensitivity and responsiveness. In starved rats, the treadmill run also enhanced the ability of insulin to stimulate these processes; however, this was solely due to an increase in insulin sensitivity. No change in insulin responsiveness was observed. The results indicate that the enhanced sensitivity of muscle to insulin after exercise is not dependent on glycogen depletion, whereas increased insulin responsiveness does not occur in its absence. They also suggest that the mechanisms by which prior exercise acts to increase insulin sensitivity and responsiveness are different.