Calsequestrin-1 (CASQ1) is a moderate-affinity, high-capacity Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) terminal cisternae of skeletal muscle. CASQ1 functions as both a Ca(2+)-binding protein and a luminal regulator of ryanodine receptor (RYR1)-mediated Ca(2+) release. Mice lacking skeletal CASQ1 are viable but exhibit reduced levels of releasable Ca(2+) and altered contractile properties. Here we report that CASQ1-null mice exhibit increased spontaneous mortality and susceptibility to heat- and anesthetic-induced sudden death. Exposure of CASQ1-null mice to either 2% halothane or heat stress triggers lethal episodes characterized by whole-body contractures, elevated core temperature, and severe rhabdomyolysis, which are prevented by prior dantrolene administration. The characteristics of these events are remarkably similar to analogous episodes observed in humans with malignant hyperthermia (MH) and animal models of MH and environmental heat stroke (EHS). In vitro studies indicate that CASQ1-null muscle exhibits increased contractile sensitivity to temperature and caffeine, temperature-dependent increases in resting Ca(2+), and an increase in the magnitude of depolarization-induced Ca(2+) release. These results demonstrate that CASQ1 deficiency alters proper control of RYR1 function and suggest CASQ1 as a potential candidate gene for linkage analysis in families with MH/EHS where mutations in the RYR1 gene are excluded.