The Ca 2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca 2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca 2+ -dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhyth-mias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca 2+ -dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca 2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca 2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca 2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca 2+ concentrations above 0.2 mM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca 2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca 2+ saturated under resting Ca 2+ conditions. Conversely, the N-domain senses Ca 2+ exactly in the transition from resting to activating Ca 2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca 2+ during Ca 2+ oscillations, while the CaM N-domain functions as a dynamic Ca 2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.