Reactive oxygen species such as superoxide (O2-) and H2O2 are produced at low levels in resting muscles and at substantially higher levels in exercising muscles. Increased respiratory activity with exercise leads to O2- production by the NADPH oxidase reaction and the subsequent generation of H2O2 from O2- by spontaneous dismutation or by the superoxide dismutase reaction. The long-lasting (24-h) depression of contractile function after exercise has been linked to damage of one or more proteins important in the excitation-contraction coupling process. We studied mechanically and chemically skinned fibers from the extensor digitorum longus muscle of the rat to evaluate the effects of a 5-min exposure to 1.0 mM H2O2 on muscle function. We found that H2O2 had no effect on the isometric force-producing properties of the contractile apparatus or on Ca2+ uptake by the sarcoplasmic reticulum. It did, however, significantly affect Ca2+ release from the sarcoplasmic reticulum. Maximum depolarization-induced Ca2+ release was inhibited, and the sensitivity to depolarization was decreased. Ca(2+)-induced release was completely blocked. We conclude that elevated levels of H2O2 with exercise are capable of damaging one or more proteins of the excitation-contraction coupling process to produce a disruption in function that can account, at least in part, for the long-lasting effects of fatiguing stimulation.