Previously we have demonstrated that PKA-dependent phosphorylation at S2809 is associated with a significant increase in RyR2 channel open probability (Po), and characteristic changes in gating¹. To further investigate RyR2 phosphorylation, sarcoplasmic reticulum vesicles were isolated² from sheep hearts obtained from an abattoir and were either incorporated into artificial membranes for single-channel studies or used for Western blot¹. In the presence of 50µM cytosolic free Ca²⁺, 5 min incubation with 5mM Mg²⁺ and 1mM ATP, followed by wash out to control conditions, significantly increased channel Po (from 0.071±0.023 to 0.334±0.076 (SEM; n=17, p<0.01, Student’s t-test)). The increase in Po however, was smaller than that observed when exogenous PKA was included in the incubation medium (from 0.126±0.035 to 0.574±0.106 (SEM; n=10). Closer examination of the individual Mg²⁺ATP treated channels suggested the presence of two different sub-populations of RyR2. In 10 of 17 channels, Mg²⁺ATP treatment resulted in an increase in channel Po and a change in channel gating similar to that observed after PKA-dependent phosphorylation (Po rose from 0.033±0.011 to 0.534±0.079 (SEM; n=10). In the remaining channels, Mg²⁺ATP had no sustained effect (n=7). ATP alone, in the absence of Mg²⁺, produced a fully reversible increase in Po in all channels treated (n=12). It is possible that the heterogeneous response of the channels to Mg²⁺ATP is due to the close association of an endogenous kinase with some of the RyR2 channels reconstituted into bilayers. Use of the PKA inhibitor, PKI (10 µM), did not prevent the Mg²⁺ATP dependent increase in channel Po although it does prevent the effects of exogenously added PKA. Similarly the Ca²⁺/calmodulin-dependent protein kinase (CaMKII) inhibitor, autocamtide-2 related inhibitory peptide II (AIPII) (50 nM), did not prevent the increase in Po at concentrations expected to inhibit CaMKII. Increasing the concentration of AIPII to a level reported to inhibit the action of PKC (1 µM) however, prevented the irreversible Mg²⁺ATP-induced changes. Further, use of the PKC specific inhibitor, chelerythrine chloride, also prevented the Mg²⁺ATP related change in channel activity. In the presence of 1µM chelerythrine chloride, Po was 0.120±0.039 before and 0.044±0.021 after treatment with Mg²⁺ATP (SEM; n=7). Chelerythrine chloride alone had no effect on channel Po. Although chelerythrine chloride inhibited the Mg²⁺ATP-dependent increase in channel Po, it did not prevent phosphorylation of RyR2 at S2809. Western blot analysis demonstrated that under lipid bilayer comparable conditions, only PKI prevented Mg²⁺ATP phosphorylation of S2809. We conclude that RyR2 channels may be associated with and phosphorylated by an endogenous kinase, possibly PKC, which does not appear to phosphorylate S2809.