The mechanism of Ca2+ release from the sarcoplasmic reticulum (SR) of slow and fast twitch muscle was compared by examining biochemical characteristics, ryanodine binding, Ca2+ efflux, and single Ca2+ channel properties of SR vesicles. Although many features of the Ca2+ release channel were comparable, two functional assays revealed remarkable differences. The comparable properties include: a high molecular weight protein from both types of muscle was immunologically equivalent, and Scatchard analysis of [3H]ryanodine binding to SR showed that the Kd was similar for slow and fast SR. In the flux assay the sensitivity to the agonists caffeine, doxorubicin, and Ca2+ and the antagonists Mg2+, ruthenium red, and tetracaine differed only slightly. When SR vesicles were incorporated into lipid bilayers, the single-channel conductances of the Ca2+ release channels were indistinguishable. The distinguishing properties are: When Ca2+ release from passively 45Ca(2+)-loaded SR were monitored by rapid filtration, the initial rates of Ca2+ release induced by Ca2+ and caffeine were three times lower in slow SR than in fast SR. Similarly, when Ca2+ release channels were incorporated into lipid bilayers, the open probability of the slow SR channel was markedly less, mainly due to a longer mean closed time. Our results indicate that slow and fast muscle have ryanodine receptors that are biochemically analogous, yet functional differences in the Ca2+ release channel may contribute to the different time to peak contraction observed in intact slow and fast muscles.