Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor ( RyR2 ) and cardiac calsequestrin ( CASQ2 ) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca 2+ -signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2 R33Q in adult rat myocytes led to an increase in excitation–contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca 2+ waves) and local Ca 2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2 WT ). As revealed by a Ca 2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca 2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca 2+ ]. Recombinant CASQ2 WT and CASQ2 R33Q exhibited similar Ca 2+ -binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca 2+ ] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca 2+ . These results show that intracellular Ca 2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.