It has previously been suggested that exercise training leads to increased whole body insulin sensitivity. However, the specific tissues and metabolic pathways involved have not been examined in vivo. By combining the euglycemic clamp with administration of glucose tracers, [3H]2-deoxyglucose (2DG), [14C]glucose, and [3H]glucose, in vivo insulin action at the whole body level and within individual tissues has been assessed in exercise-trained (ET, running 1 h/d for 7 wk) and sedentary control rats at four insulin doses. Whole body insulin sensitivity was significantly increased in ET. In addition, the skeletal muscles, soleus, red and white gastrocnemius, extensor digitorum longus (EDL), and diaphragm all showed increased sensitivity of insulin-stimulated 2DG uptake with training. With the exception of EDL, no significant difference in insulin-mediated glycogen synthesis between control and ET could be found. Therefore, the increased insulin-induced 2DG uptake observed in muscle following training is apparently directed towards glucose oxidation. In ET animals, adipose tissue exhibited a significant increase in insulin-mediated 2DG uptake and [14C]glucose incorporation into free fatty acids but there was no difference from control in any parameters measured in lung or liver. EDL and white gastrocnemius, which are not primarily involved during exercise of this type, also demonstrated increased insulin sensitivity following training. In conclusion, exercise training results in a marked increase in whole body insulin sensitivity related mainly to increased glucose oxidation in skeletal muscle. This effect may be mediated by systemic as well as local factors and is likely to be of therapeutic value in pathological conditions exhibiting insulin resistance.