The solubilized [3H]ryanodine receptor from cardiac sarcoplasmic reticulum was centrifuged through linear sucrose gradients. A single peak of radioactivity with apparent sedimentation coefficient of approximately 30S specifically comigrated with a high molecular weight protein of apparent relative molecular mass approximately 400,000. Incorporation of the ryanodine receptor into lipid bilayers induced single Ca2+ channel currents with conductance and kinetic behavior almost identical to that of native cardiac Ca2+ release channels. These results suggest that the cardiac ryanodine receptor comprises the Ca2+ release channel involved in excitation-contraction coupling in cardiac muscle.