To evaluate the relationship between enhanced insulin action and level of exercise training, in vivo glucose uptake was assessed in the absence of added insulin and during insulin-stimulated conditions for three activity levels of voluntarily trained rats (low 2-5 km/day, medium 6-9 km/day, high 11-16 km/day). After rats rested for 24 h and fasted overnight, glucose uptake was estimated by comparing steady-state serum glucose (SSSG) levels at low insulin (SSSI) concentrations achieved during an insulin suppression test. In the absence of added insulin, SSSI averaged approximately 20 microU/ml and glucose uptake was similar for high runners and younger weight-matched controls. However, with insulin added to sustain SSSI at approximately 35 microU/ml, SSSG was significantly reduced in all runners (P less than 0.02), with the lowest value attained in high runners. Fasting serum triglycerides were also reduced in all runners (P less than 0.05), with the lowest values seen in medium and high runners. The concentration of glycogen in liver and select skeletal muscles at the start of the study was not different between trained and control rats, suggesting that enhanced insulin-stimulated glucose uptake was not the result of lower glycogen levels. In addition, glycogen synthase and succinate dehydrogenase activities in biceps femoris muscle were only elevated for high runners, but glycogen synthase activity was not enhanced in plantaris muscle and was decreased in soleus muscle. These findings indicate that enhanced insulin-stimulated glucose uptake and reduced serum triglyceride concentrations induced in exercise-trained rats at varying activity levels are dissociated from changes in glycogen synthase and oxidative enzyme activity for skeletal muscle.