The 5′AMP-activated protein kinase (AMPK) is stimulated by contractile activity in rat skeletal muscle. AMPK has emerged as an important signaling intermediary in the regulation of cell metabolism being linked to exercise-induced changes in muscle glucose and fatty acid metabolism. In the present study, we determined the effects of exercise on isoform-specific AMPK activity (α1 and α2) in human skeletal muscle. Needle biopsies of vastus lateralis muscle were obtained from seven healthy subjects at rest, after 20 and 60 min of cycle ergometer exercise at 70% of VO₂max, and 30 min following the 60 min exercise bout. In comparison to the resting state, AMPK α2 activity significantly increased at 20 and 60 min of exercise, and remained at a higher level with 30 min of recovery. AMPK α1 activity tended to slightly decrease with 20 min of exercise at 70%VO₂max; however, the change was not statistically significant. AMPK α1 activities were at basal levels at 60 min of exercise and 30 min of recovery. On a separate day, the same subjects exercised for 20 min at 50% of VO₂max. Exercise at this intensity did not change α2 activity, and similar to exercise at 70% of VO₂max, there was no significant change in α1 activity. In conclusion, exercise at a higher intensity for only 20 min leads to increases in AMPK α2 activity but not α1 activity. These results suggest that the α2-containing AMPK complex, rather than α1, may be involved in the metabolic responses to exercise in human skeletal muscle.