The purpose of this study was to determine if changes in oxidative stress biomarkers in blood and skeletal muscles are similar in normal and antioxidant supplemented rats after a downhill run. Sixty-six male Sprague-Dawley rats were pretreated with a normal rat diet or diet + antioxidants (2,000 mg vitamin C + 1,000 IU vitamin E/kg diet) for 2 weeks. Exercised rats ran 90 min on a rodent treadmill at a speed of 16 m/min at -16 degrees grade. Rats were sacrificed either at rest, immediately, 2 hrs, or 48 hrs postexercise. Malondialdehyde (MDA) and protein carbonyl (PC) concentrations and glutathione status in blood, vastus lateralis (white fast-twitch), vastus intermedius (red fast-twitch), and soleus (slow-twitch) muscles were determined. A significant increase from rest in PC occurred in plasma, vastus intermedius and soleus muscle 2 hrs after the downhill run (p < 0.05), with no changes observed at any other times postexercise. Antioxidant supplementation significantly decreased PC concentrations in both vastus intermedius and soleus muscles at all times combined (p < 0.05). MDA and glutathione status in blood and muscles were unaffected by either the downhill run or antioxidant treatment. For PC and MDA, the concentrations were lower in blood as compared to skeletal muscle, with the opposite finding for oxidized glutathione; however, the pattern of response postexercise was similar. These data indicate that (a) PC, but not MDA or oxidized glutathione, is elevated transiently following downhill running in male rats; (b) the elevation in PC postexercise occurs in plasma, vastus intermedius, and soleus muscles; (c) antioxidant therapy can attenuate PC in vastus intermedius, and soleus muscles; and (d) while the concentrations of oxidative stress biomarkers differ between blood and the various skeletal muscles, the pattern of response postexercise is similar.