Properties of the sarcoplasmic reticulum (SR) with respect to Ca2+ loading and release were measured in mechanically skinned fiber preparations from isolated extensor digitorum longus (EDL) muscles of the rat that were either kept at room temperature (23°C) or exposed to temperatures in the upper physiological range for mammalian skeletal muscle (30 min at 40 or 43°C). The ability of the SR to accumulate Ca2+ was significantly reduced by a factor of 1.9–2.1 after the temperature treatments due to a marked increase in SR Ca2+ leak, which persisted for at least 3 h after treatment. Results with blockers of Ca2+ release channels (ruthenium red) and SR Ca2+ pumps [2,5-di( tert-butyl)-1,4-hydroquinone] indicate that the increased Ca2+ leak was not through the SR Ca2+ release channel or the SR Ca2+ pump, although it is possible that the leak pathway was via oligomerized Ca2+ pump molecules. No significant change in the maximum SR Ca2+-ATPase activity was observed after the temperature treatment, although there was a tendency for a decrease in the SR Ca2+-ATPase. The observed changes in SR properties were fully prevented by the superoxide (O2•−) scavenger Tiron (20 mM), indicating that the production of O2•− at elevated temperatures is responsible for the increase in SR Ca2+ leak. Results show that physiologically relevant elevated temperatures 1) induce lasting changes in SR properties with respect to Ca2+ handling that contribute to a marked increase in the SR Ca2+ leak and, consequently, to the reduction in the average coupling ratio between Ca2+ transport and SR Ca2+-ATPase and muscle performance, and 2) that these changes are mediated by temperature-induced O2•− production.