1. The effect on sarcoplasmic reticulum (SR) function of exposure to high intracellular [Ca2+] was studied in mechanically skinned fibres from the extensor digitorum longus muscle of the rat, using caffeine to assay the SR Ca2+ content. 2. A 15 s exposure to 50 microM Ca2+ irreversibly reduced the ability of the SR to load/retain Ca2+ and completely abolished depolarization-induced Ca2+ release, whereas a 90 s exposure to 10 microM Ca2+ had no detectable effect on either function. The reduction in net SR Ca2+ uptake: (a) was near-maximal with treatment at 50 microM Ca2+, (b) was unrelated to voltage-sensor function, and (c) persisted unchanged for > 20 min. The reduction was primarily due to a threefold increase in leakage of Ca2+ out of the SR. This increased leakage was not substantially blocked by the presence of 10 mM Mg2+ or 2 microM Ruthenium Red. 3. The adverse effect on SR function of exposure to high [Ca2+] could also be observed by the reduction in the ability of the SR to maintain a low [Ca2+] within the skinned fibre in the face of elevated [Ca2+] in the bathing solution. When bathed in a solution with approximately 1.5 microM Ca2+ (0.75 mM CaEGTA-EGTA), skinned fibres produced only low force responses for many minutes, but after high [Ca2+] treatment (15 s exposure to 50 microM Ca2+) they showed large, steady or oscillatory force responses. 4. These findings indicate that, in addition to uncoupling the Ca2+ release channels from the voltage sensors, exposure of skinned fibres to high [Ca2+] causes a persistent increase in resting Ca2+ efflux from the SR. Such efflux in an intact fibre would alter the distribution of Ca2+ between the SR, the cytoplasm and the extracellular solution. These results may be relevant to the basis of low-frequency fatigue and possibly other conditions in muscle.