The effects of endurance training on the response of muscle AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) to moderate treadmill exercise were examined. In red quadriceps, there was a large activation of alpha 2-AMPK and inactivation of ACC in response to exercise. This response was greatly reduced after training, probably because of reduced metabolic stress. In white quadriceps, there were no effects of exercise on AMPK or ACC, but alpha 2-activity was higher after training because of increased phosphorylation of Thr(172). In soleus, there were small increases in alpha 2-activity during exercise that were not affected by training. The expression of all seven AMPK subunit isoforms was also examined. The beta 2- and gamma 2-isoforms were most highly expressed in white quadriceps, and gamma 3 was expressed in red quadriceps and soleus. There was a threefold increase in expression of gamma 3 after training in red quadriceps only. Our results suggest that gamma 3 might have a special role in the adaptation to endurance exercise in muscles utilizing oxidative metabolism.