We aimed to define the relative contribution of both PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during β-adrenergic receptor (βAR) stimulation. Rat hearts were perfused with increasing concentrations of the β-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca2+ influx to the cell by low [Ca]o plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [3H]-ryanodine binding and fast Ca2+ release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca2+. Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under βAR stimulation by PKA and CaMKII, respectively. The isoproterenolinduced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the β-induced increase in the channel activity as indicated by the enhancement of the [3H]-ryanodine binding and the velocity of fast SR Ca2+ release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca2+ release during βAR stimulation.Centro de Investigaciones Cardiovasculares