Previous studies have reported that oxidizing free radical species are generated during exercise, and there has been considerable interest in the potential effects of these on exercising tissues. We hypothesized that contracting skeletal muscle was a major source of oxidizing free radical species and that untrained skeletal muscle would adapt to the oxidative stress of a single short period of contractile activity by upregulation of the activity of cytoprotective proteins in the absence of overt cellular damage. Fifteen minutes of aerobic contractile activity was found to induce a rapid release of superoxide anions from mouse skeletal muscle in vivo, and studies with contracting cultured skeletal muscle myotubes confirmed that this was due to release from myocytes rather than other cell types present within muscle tissue in vivo. This increased oxidant production caused a rapid, transient reduction in muscle protein thiol content, followed by increases in the activities of superoxide dismutase and catalase and in content of heat shock proteins. These changes occurred in the absence of overt damage to the muscle cells.