Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca²⁺ and inactivated by higher (~1 mM) Ca²⁺. To gain insight into a mechanism underlying Ca²⁺-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 [³H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca²⁺-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W and R4736Q) in the cytoplasmic loop between S2 and S3 transmembrane segments (S2-S3 loop) greatly reduced Ca²⁺-dependent channel inactivation. Activities of these mutant channels were suppressed at 10-100 µM Ca²⁺, and the suppressions were relieved by 1 mM Mg²⁺. The Ca²⁺- and Mg²⁺-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 Ca²⁺ affinities for channel activation and decreased Ca²⁺ affinities for inactivation, but impairment of Ca²⁺-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 Ca²⁺-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 Ca²⁺ and Mg²⁺. Four malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop conferred RyR2-type Ca²⁺- and Mg²⁺-dependent channel regulation.