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 (alpha 1 and alpha 2) 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 VO(2)max, and 30 min following the 60 min exercise bout. In comparison to the resting state, AMPK alpha 2 activity significantly increased at 20 and 60 min of exercise, and remained at a higher level with 30 min of recovery. AMPK al activity tended to slightly decrease with 20 min of exercise at 70%VO(2)max; however, the change was not statistically significant. AMPK alpha 1 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 VO(2)max. Exercise at this intensity did not change alpha 2 activity, and similar to exercise at 70% of VO(2)max, there was no significant change in alpha 1 activity. In conclusion, exercise at a higher intensity for only 20 min leads to increases in AMPK alpha 2 activity but not alpha 1 activity. These results suggest that the alpha 2-containing AMPK complex, rather than al, may be involved in the metabolic responses to exercise in human skeletal muscle.