Endurance exercise induces increases in mitochondrial biogenesis, GLUT4 expression, and insulin action in skeletal muscle. It has been reported that supplementation with the antioxidant vitamins C and E prevents these adaptations. PURPOSE: The purpose of this study was to reevaluate the effects these antioxidants on the adaptive response of mitochondria, GLUT4 and insulin action to endurance exercise. METHODS: Male Wistar rats were assigned to 4 groups: Vehicle-Sedentary (Ve-Sed), Vehicle-Exercise (Ve-Ex), Anti-Oxidant sedentary (AO-Sed), and Anti-Oxidant-Exercise (AO-EX). The rats in the antioxidant groups were given 750 mg vitamin C and 150 mg vitamin E once daily by stomach tube for 7 days. The rats in the exercise groups were exercised by swimming for the last 3 days of the 7 day antioxidant treatment period. Immediately after last bout of exercise, blood was collected for TBARS analysis. The rats were killed 18 hr after exercise. RESULTS: Immediately after exercise, plasma TBARS were elevated in the Ve-Ex but not in the AO-Ex group providing evidence for an antioxidant effect (Ve-Sed; 0.55±0.04, Ve-Ex; 0.83±0.06, AO-Sed; 0.54±0.02, AO-Ex; 0.57±0.04 μM). There was a large increase in insulin-responsiveness of glucose in the epitrochlearis muscles of the exercised animals transport (Ve-Sed; 2.28±0.22, Ve-Ex; 5.80±0.74, AO-Sed; 2.81±0.39, AO-Ex; 5.45±0.96 μmol/ml/20 min) that was mediated by a ∼2-fold increase in GLUT4 expression (Ve-Sed; 1.00±0.1, Ve-Ex; 2.70±0.18, AO-Sed; 1.05±0.12, AO-Ex; 2.64±0.2). The levels of PGC-1· and a number of mitochondrial enzyme proteins (COXI, COXIV, NADHUO, SUO and SOD) were increased about 2-fold in triceps muscle in response to the exercise. There was no difference in the magnitudes of these adaptive responses between the Ve-Ex and AO-Ex groups. CONCLUSION: These results show that very large doses of antioxidant vitamins do not prevent the exercise-induced adaptive responses of muscle mitochondria, GLUT4 or insulin action to exercise. Supported by NIH Grant AG000425. K.H was supported by Research Fellow of the Japan Society for the Promotion of Science.