Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, induces necrosis in skeletal muscle. In isolated organelles, it has been reported that myotoxin a reduces Ca2+ uptake into the sarcoplasmic reticulum. The effects of the toxin on Ca2+ regulation were examined in heavy sarcoplasmic reticulum fractions from human and equine skeletal muscle. Ca2+ uptake and release (the threshold of Ca(2+)-induced Ca2+ release) were examined by dual wavelength spectrophotometry. The toxin lowered the threshold of Ca(2+)-induced Ca2+ release in a dose-dependent manner (1-10 microM) and this effect was antagonized by ruthenium red, a Ca2+ release channel blocker. Ca2+ uptake into equine heavy sarcoplasmic reticulum was not decreased by myotoxin a (10 microM) when Ca2+ release was blocked by ruthenium red. [3H]Ryanodine binding to equine heavy sarcoplasmic reticulum was converted from a relatively low affinity state to a higher affinity state by myotoxin a. These results suggest that the dominant effect of myotoxin a is to increase the Ca2+ sensitivity for the opening of the calcium release channel (ryanodine receptor). Myotoxin a may prove to be a useful tool to probe the modulation of calcium release in sarcoplasmic reticulum fractions.