Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca and inactivated by higher (∼1 mM) Ca To gain insight into a mechanism underlying Ca-dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy-associated mutations and determined RyR1 channel activities by [H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W, and R4736Q) in the cytoplasmic loop between the S2 and S3 transmembrane segments (S2-S3 loop) greatly reduced Ca-dependent channel inactivation. Activities of these mutant channels were suppressed at 10-100 μM Ca, and the suppressions were relieved by 1 mM Mg The Ca- and Mg-dependent regulation of S2-S3 loop RyR1 mutants are similar to those of the cardiac isoform of RyR (RyR2) rather than wild-type RyR1. Two mutations (T4825I and H4832Y) in the S4-S5 cytoplasmic loop increased Ca affinities for channel activation and decreased Ca affinities for inactivation, but impairment of Ca-dependent inactivation was not as prominent as those of S2-S3 loop mutants. Three mutations (T4082M, S4113L, and N4120Y) in the EF-hand domain showed essentially the same Ca-dependent channel regulation as that of wild-type RyR1. The results suggest that nine RyR1 mutants associated with skeletal muscle diseases were differently regulated by Ca and Mg Four malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop conferred RyR2-type Ca- and Mg-dependent channel regulation.