The functional effects of calmodulin (CaM) on single cardiac sarcoplasmic reticulum Ca²⁺ release channels (ryanodine receptors) (RyR2s) were determined in the presence of two endogenous channel effectors, MgATP and reduced glutathione, using the planar lipid bilayer method. Single-channel activities, number of events, and open and close times were determined at varying cytosolic Ca²⁺ concentrations. CaM reduced channel open probability at <10μM Ca²⁺ by decreasing channel events and mean open times and increasing mean close times. At >10μM Ca²⁺, CaM was less effective in inhibiting RyR2. CaM decreased mean open times but increased channel events, without significantly affecting mean close times. A series of voltage pulses was applied to the bilayer from +50 to −50mV and from −50mV to +50mV to rapidly increase and decrease open channel-mediated sarcoplasmic reticulum lumenal to cytosolic Ca²⁺ fluxes. CaM decreased the duration of the open events after the voltage switch from −50mV to +50mV. In parallel experiments, a Ca²⁺-insensitive calmodulin mutant was without effect on RyR2 activity. The results are discussed in terms of a possible role of CaM in the termination of cardiac sarcoplasmic reticulum Ca²⁺ release.