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 (30min at 40°C 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 hours after treatment. Results with blockers of Ca²⁺-release channels (Ruthenium Red) and SR Ca²⁺-pumps (2,5-di(tert-butyl)-1,4-hydroquinone (TBQ)), 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 oligomerised 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 (20mM) 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 (i) induce lasting changes in SR properties with respect to Ca²⁺-handling which 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 (ii) that these changes are mediated by temperature-induced O₂^- production.