To find out whether the decrease in muscle performance of isolated mammalian skeletal muscle associated with the increase in temperature toward physiological levels is related to the increase in muscle superoxide (O2•−) production, O2•− released extracellularly by intact isolated rat and mouse extensor digitorum longus (EDL) muscles was measured at 22, 32, and 37°C in Krebs-Ringer solution, and tetanic force was measured in both preparations at 22 and 37°C under the same conditions. The rate of O2•− production increased marginally when the temperature was increased from 22 to 32°C, but increased fivefold when the temperature was increased from 22 to 37°C in both rat and mouse preparations. This increase was accompanied by a marked decrease in tetanic force after 30 min incubation at 37°C in both rat and mouse EDL muscles. Tetanic force remained largely depressed after return to 22°C for up to 120 min. The specific maximum Ca2+-activated force measured in mechanically skinned fibers after the temperature treatment was markedly depressed in mouse fibers but was not significantly depressed in rat muscle fibers. The resting membrane and intracellular action potentials were, however, significantly affected by the temperature treatment in the rat fibers. The effects of the temperature treatment on tetanic force, maximum Ca2+-activated force, and membrane potential were largely prevented by 1 mM Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a membrane-permeable superoxide dismutase mimetic, indicating that the increased O2•− production at physiological temperatures is largely responsible for the observed depression in tetanic force at 37°C by affecting the contractile apparatus and plasma membrane.