Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca] and intra-SR [Ca] were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca spark frequency approximately two-fold. This effect was associated with an increase in SR Ca load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca leak.