To examine the thermal instability and the role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR) Ca2+-ATPase function, crude homogenates were prepared from the white portion of the gastrocnemius (WG) adult rat muscles ( n = 9) and incubated in vitro for ≤60 min either at a normal resting body temperature (37°C) or at a temperature indicative of exercise-induced hyperthermia (41°C) with DTT and without DTT (CON). In general, treatment with DTT resulted in higher Ca2+-ATPase and Ca2+ uptake values (nmol · mg protein−1 · min−1, P < 0.05), an effect that was not specific to time of incubation. Incubations at 41°C resulted in lower ( P< 0.05) Ca2+ uptake rates (156 ± 18 and 35.9 ± 3.3) compared with 37°C (570 ± 54 and 364 ± 26) at 30 and 60 min, respectively. At 37°C, ryanodine (300 μM), which was used to block Ca2+ release from the calcium release channel, prevented the time-dependent decrease in Ca2+ uptake. A general inactivation ( P < 0.05) of maximal Ca2+-ATPase activity ( V max) in CON was observed with incubation time (0 > 30 > 60 min), with the effect being more pronounced ( P < 0.05) at 41°C compared with 37°C. The Hill slope, a measure of co-operativity, and the pCa50, the cytosolic Ca2+ concentration required for half-maximal activation of Ca2+-ATPase activity, decreased ( P < 0.05) at 41°C only. Treatment with DTT attenuated the alterations in enzyme kinetics. The increase in V max with the Ca2+ionophore A-23187 was less pronounced at 41°C compared with 37°C. It is concluded that exposure of homogenates to a temperature typically experienced in exercise results in a reduction in the coupling ratio, which is mediated primarily by lower Ca2+ uptake and occurs as a result of increases in membrane permeability to Ca2+. Moreover, the decreases in Ca2+-ATPase kinetics in WG with sustained heat stress result from SH oxidation.