Myotoxin alpha from prairie rattlesnakes induced Ca2+ release from the heavy fraction of sarcoplasmic reticulum at submicromolar concentrations. 125I-Labeled myotoxin alpha (125I-myotoxin alpha) specifically bound to the heavy fraction. Fractionation of the solubilized heavy fraction with a spermine-agarose column gave purified calsequestrin as a major binding protein of 125I-myotoxin alpha. We have first indicated that calsequestrin is a target protein for Ca(2+)-releasing action of myotoxin alpha. Calsequestrin probably plays a key role in physiological Ca2+ release from sarcoplasmic reticulum.