There is evidence in rodents that Ca2+-calmodulin-dependent protein kinase II (CaMKII) activity is higher in contracting skeletal muscle, and this kinase may regulate skeletal muscle function and metabolism during exercise. To investigate the effect of exercise on CaMKII in human skeletal muscle, healthy men (n = 8) performed cycle ergometer exercise for 40 min at 76 ± 1% peak pulmonary O2 uptake (·VO2peak), with skeletal muscle samples taken at rest and after 5 and 40 min of exercise. CaMKII expression and activities were examined by immunoblotting and in vitro kinase assays, respectively. There were no differences in maximal (+ Ca2+, CaM) CaMKII activity during exercise compared with rest. Autonomous (-Ca2+, CaM) CaMKII activity was 9 ± 1% of maximal at rest, remained unchanged at 5 min, and increased to 17 ± 1% (P < 0.01) at 40 min. CaMKII autophosphorylation at Thr287 was 50-70% higher during exercise, with no differences in CaMKII expression. The effect of maximal aerobic exercise on CaMKII was also examined (n = 9), with 0.7-to 1.5-fold increases in autonomous CaMKII activity, but no change in maximal CaMKII activity. CaMKIV was not detected in human skeletal muscle. In summary, exercise increases the activity of CaMKII in skeletal muscle, suggesting that it may have a role in regulating skeletal muscle function and metabolism during exercise in humans.