This study tested the hypothesis that reactive oxygen intermediates present in unfatigued skeletal muscle act to enhance contractile function. Fiber bundles from rat diaphragm were incubated with exogenous catalase (an antioxidant enzyme that dehydrates hydrogen peroxide to molecular oxygen and water) to decrease the tissue concentration of reactive oxygen intermediates. Catalase (10(3) U/ml) significantly decreased twitch characteristics (time to peak tension, half-relaxation time, peak force, and twitch-to-tetanus force ratio), thereby shifting the force-frequency relationship to the right. Catalase effects were dose dependent. Concentrations of 1 to 10(5) U/ml progressively depressed submaximal (30-Hz) tetanic stress, whereas concentrations > 10(5) U/ml were toxic, inhibiting maximal (200-Hz) tetanic stress (P < 0.0001). Exogenous hydrogen peroxide (10(-4) to 10(-2)M) increased peak twitch stress (P < 0.03) and lengthened both time to peak tension (P < 0.02) and half-relaxation time (P < 0.02). Selective removal of superoxide anion radicals with the use of superoxide dismutase produced dose-dependent contractile inhibition similar to that produced by catalase. We conclude that the reactive oxygen intermediates present in unfatigued skeletal muscle have a positive effect on excitation-contraction coupling and are obligatory for optimal contractile function.