The predominant cardiac Ca²⁺/calmodulin-dependent protein kinase (CaMK) is CaMKIIδ. Here we acutely overexpress CaMKIIδ_C using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKIIδ_C transgenic mice. CaMKIIδ_C protein expression and activation state (autophosphorylation) were increased 5- to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKIIδ_C versus LacZ expressing myocytes. However, the contraction–frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced (τ_0.5Hz/τ_3Hz=2.14±0.10 versus 1.87±0.10), and peak Ca²⁺ current (I_Ca) was increased by 31% (−7.1±0.5 versus −5.4±0.5 pA/pF, P<0.05). Ca²⁺ transient amplitude was not significantly reduced (−27%, P=0.22), despite dramatically reduced sarcoplasmic reticulum (SR) Ca²⁺ content (41%; P<0.05). Thus fractional SR Ca²⁺ release was increased by 60% (P<0.05). Diastolic SR Ca²⁺ leak assessed by Ca²⁺ spark frequency (normalized to SR Ca²⁺ load) was increased by 88% in CaMKIIδ_C versus LacZ myocytes (P<0.05; in an multiplicity-of-infection–dependent manner), an effect blocked by CaMKII inhibitors KN-93 and autocamtide-2–related inhibitory peptide. This enhanced SR Ca²⁺ leak may explain reduced SR Ca²⁺ content, despite measured levels of SR Ca²⁺-ATPase and Na⁺/Ca²⁺ exchange expression and function being unaltered. Ryanodine receptor (RyR) phosphorylation in CaMKIIδ_C myocytes was increased at both Ser2809 and Ser2815, but FKBP12.6 coimmunoprecipitation with RyR was unaltered. This shows for the first time that acute CaMKIIδ_C overexpression alters RyR function, leading to enhanced SR Ca²⁺ leak and reduced SR Ca²⁺ content but without reducing twitch contraction and Ca²⁺ transients. We conclude that this is attributable to concomitant enhancement of fractional SR Ca²⁺ release in CaMKIIδ_C myocytes (ie, CaMKII-dependent enhancement of RyR Ca²⁺ sensitivity during diastole and systole) and increased I_Ca.