Previous studies in transgenic mice and with isolated ryanodine receptors (RyR) have indicated that Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR and activate local diastolic sarcoplasmic reticulum (SR) Ca²⁺ release events (Ca²⁺ sparks) and RyR channel opening. Here we use relatively controlled physiological conditions in saponin-permeabilized wild type (WT) and phospholamban knockout (PLB-KO) mouse ventricular myocytes to test whether exogenous preactivated CaMKII or endogenous CaMKII can enhance resting Ca²⁺ sparks. PLB-KO mice were used to preclude ancillary effects of CaMKII mediated by phospholamban phosphorylation. In both WT and PLB-KO myocytes, Ca²⁺ spark frequency was increased by both preactivated exogenous CaMKII and endogenous CaMKII. This effect was abolished by CaMKII inhibitor peptides. In contrast, protein kinase A catalytic subunit also enhanced Ca²⁺ spark frequency in WT, but had no effect in PLB-KO. Both endogenous and exogenous CaMKII increased SR Ca²⁺ content in WT (presumably via PLB phosphorylation), but not in PLB-KO. Exogenous calmodulin decreased Ca²⁺ spark frequency in both WT and PLB-KO (K_0.5 ≈100 nmol/L). Endogenous CaMKII (at 500 nmol/L [Ca²⁺]) phosphorylated RyR as completely in <4 minutes as the maximum achieved by preactivated exogenous CaMKII. After CaMKII activation Ca²⁺ sparks were longer in duration, and more frequent propagating SR Ca²⁺ release events were observed. We conclude that CaMKII-dependent phosphorylation of RyR by endogenous associated CaMKII (but not PKA-dependent phosphorylation) increases resting SR Ca²⁺ release or leak. Moreover, this may explain the enhanced SR diastolic Ca²⁺ leak and certain triggered arrhythmias seen in heart failure.