Two isoforms of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor or RYR) are expressed together in the skeletal muscles of most vertebrates. We have studied physiological properties of the two isoforms (alpha and beta) by comparing SR preparations from specialized fish muscles that express the alpha isoform alone to preparations from muscles containing both alpha and beta. Regulation of channel activity was assessed through [3H]ryanodine binding and reconstitution into planar lipid bilayers. Distinct differences were observed in the calcium-activation and -inactivation properties of the two isoforms. The fish alpha isoform, expressed alone in extraocular muscles, closely resembled the rabbit skeletal muscle RYR. Maximum [3H]ryanodine binding and maximum open probability (Po) of the alpha RYR were achieved from 1 to 10 microM free Ca2+. Millimolar Ca2+ reduced [3H]ryanodine binding and Po close to zero. The beta isoform more closely resembled the fish cardiac RYR in Ca2+ activation of [3H]ryanodine binding. The most prominent difference of the beta and cardiac isoforms from the alpha isoform was the lack of inactivation of [3H]ryanodine binding and Po by millimolar free Ca2+. Differences in activation of [3H]ryanodine binding by adenine nucleotides and inhibition by Mg2+ suggest that the beta and cardiac RYRs are not identical, however. [3H]ryanodine binding by the alpha RYR was selectively inhibited by 100 microM tetracaine, whereas cardiac and beta RYRs were much less affected. Tetracaine can thus be used to separate the properties of the alpha and beta RYRs in preparations in which both are present. The distinct physiological properties of the alpha and beta RYRs that are present together in most vertebrate muscles support models of EC coupling incorporating both directly coupled and Ca(2+)-coupled channels within a single triad junction.