Dantrolene is a skeletal muscle relaxant which acts by inhibiting intracellular Ca²⁺ release from sarcoplasmic reticulum (SR). It is used primarily in the treatment of malignant hyperthermia (MH), a pharmacogenetic sensitivity to volatile anesthetics resulting in massive intracellular Ca²⁺ release. Determination of the site and mechanism of action of dantrolene should contribute to the understanding of the regulation of intracellular Ca²⁺ release in skeletal muscle. Photoaffinity labeling of porcine SR with [³H]azidodantrolene, a photoactivatable analogue of dantrolene, has identified a 160 kDa SR protein with immunologic cross-reactivity to skeletal muscle ryanodine receptor (RyR) as a possible target [Palnitkar et al. (1999) J. Med. Chem. 42, 1872−1880]. Here we demonstrate specific, AMP-PCP-enhanced, [³H]azidodantrolene photolabeling of both the RyR monomer and a 160 or 172 kDa protein in porcine and rabbit SR, respectively. The 160/172 kDa protein is shown to be the NH₂-terminus of the RyR cleaved from the monomer by an endogenous protease activity consistent with that of n-calpain. MALDI-mass spectrometric analysis of the porcine 160 kDa protein identifies it as the 1400 amino acid NH₂-terminal fragment of the skeletal muscle RyR reportedly generated by n-calpain [Shevchenko et al. (1998) J. Membr. Biol. 161, 33−34]. Immunoprecipitation of solubilized, [³H]azidodantrolene-photolabeled SR protein reveals that the cleaved 160/172 kDa protein remains associated with the C-terminal, 410 kDa portion of the RyR. [³H]Dantrolene binding to both the intact and the n-calpain-cleaved channel RyR is similarly enhanced by AMP-PCP. n-Calpain cleavage of the RyR does not affect [³H]dantrolene binding in the presence of AMP-PCP, but depresses drug binding in the absence of nucleotide. These results demonstrate that the NH₂-terminus of the RyR is a molecular target for dantrolene, and suggest a regulatory role for both n-calpain activity and ATP in the interaction of dantrolene with the RyR in vivo.