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[³H]Ryanodine binds with high affinity to saturable and Ca²⁺-dependent sites in heavy sarcoplasmic reticulum (SR) preparations from rabbit skeletal and cardiac muscle. Ruthenium red, known to interfere with Ca²⁺-induced Ca²⁺ release from SR vesicles, inhibits [³H]ryanodine specific binding in both skeletal and cardiac preparations whereas Mg²⁺, Ba²⁺, Cd²⁺ and La³⁺ selectively inhibit the skeletal preparation. The toxicological relevance of the [³H]ryanodine binding site is established by the correlation of binding inhibition with toxicity for seven ryanoids including two botanical insecticides. These findings provide direct evidence for Ca²⁺-ryanodine receptor complexes that may play a role in excitation-contraction coupling.