Although the precise mechanisms underlying the dysfunction of sarcoplasmic reticulum (SR) that occurs during skeletal muscle fatigue remain obscure, it has been hypothesized that it may be attributable to oxidation of critical sulfhydryl groups residing in SR Ca²⁺-ATPase protein by endogenously produced reactive oxygen species. In order to test this hypothesis, SR Ca²⁺-ATPase activities in the absence or presence of the disulfide reducing agent, dithiothreitol (DTT), were examined in muscle homogenates of the soleus muscles (SQL) and the superficial portions of the vastus lateralis muscles (VS) from the rat subjected to exhaustive running at 50 m/min on a 10% grade. Immediately after exercise, the catalytic activity of SR Ca²⁺-ATPase was significantly depressed in VS, but not in SQL. The loss of SR Ca²⁺-ATPase activity observed in VS was fully recovered after treatment with DTT (1 mM) . These recovery effects of a potent disulfide reducing agent suggest that important proteins of SR Ca²⁺-ATPase may be oxidized during high-intensity exercise and that the onset of muscular fatigue may be delayed by the improved function of the cellular antioxidant