We studied whether hydrogen peroxide (H(2)O(2)) at </=10 microM activates the ryanodine receptor and decreases releasable Ca(2+) content in the sarcoplasmic reticulum after fatigue. Exposure of rabbit or frog skeletal muscle ryanodine receptors to 10 microM H(2)O(2) enhanced channel activity in lipid bilayers when the redox potential was defined at cis = -220 mV and trans = -180 mV. Channel activation by 10 microM H(2)O(2) was also observed when cis potential was set at -220 mV without defining trans potential, but the effect was less. Reduction of trans redox potential from -180 to -220 mV did not alter channel activity. H(2)O(2) at 500 microM failed to activate the channel when the redox potential was not controlled. Stimulation of the frog muscle fiber for 2 min (50 Hz, a duty cycle of 200 ms/s) decreased tetanus tension by approximately 50%. After 1 min, tetanus recovered rapidly to approximately 70% of control and thereafter slowly approached the control level. Amplitudes of caffeine- and 4-chloro-m-cresol-induced contractures were decreased after a 60-min rest. The decrease is not enhanced by exposure to 10 microM H(2)O(2). These results suggest that H(2)O(2) markedly activates the ryanodine receptor under the redox control in vitro, but externally applied H(2)O(2) may not play an important role in the postfatigue recovery process.