Ryanodine receptors/Ca2+ release channels play an important role in regulating the intracellular free calcium concentrations in both muscle and nonmuscle cells. Ryanodine, a neutral plant alkaloid, specifically binds to and modulates these Ca2+ release channels. In the work described here, we characterize the interaction of a tritium-labeled, photoactivable derivative of ryanodine (3H-labeled 10-O-[3-(4-azidobenzamido)propionyl]ryanodine ([3H]ABRy)) with the ryanodine receptor of skeletal, cardiac, and brain membranes. Scatchard analysis demonstrates that this ligand binds to a single class of high affinity sites in skeletal muscle triads. Furthermore, competition binding assays of [3H]ryanodine with skeletal, cardiac, and brain membranes in the presence of increasing concentrations of unlabeled ABRy illustrate that this azido derivative of ryanodine is able to specifically displace [3H]ryanodine from its binding site(s). Analysis of the effects of Ca2+, ATP, and KCl on [3H]ABRy binding in triad membranes shows a similar modulation of binding to that seen in these membranes with [3H]ryanodine. Photoaffinity labeling of triads with [3H]ABRy resulted in specific and covalent incorporation of [3H]ABRy into a 565-kDa protein that was shown to be the skeletal muscle ryanodine receptor. Digestion of the labeled ryanodine receptor revealed a [3H]ABRy-labeled 76-kDa tryptic fragment that was identified with an antibody directed against the COOH-terminal of the receptor. These results demonstrate that the 76-kDa COOH-terminal tryptic fragment contains the high affinity binding site for ryanodine.