Protein kinase A (PKA)-dependent phosphorylation of the cardiac ryanodine receptor (RyR) is a controversial subject; some groups report an increase in single-channel open probability (P_o) (1), while others report a reduction in P_o and [³H]ryanodine binding (2). We have therefore investigated how the degree of phosphorylation at serine 2809 (S2809) is related to the single-channel function of the sheep cardiac RyR. Sarcoplasmic reticulum membrane vesicles were isolated (4) from sheep hearts obtained from an abattoir and were either incorporated into artificial membranes for single-channel studies or were used for [³H]ryanodine binding or Western blotting. All data presented, unless stated otherwise, are mean ± SEM, and statistical significance was determined using a paired t test (n≥4). Cardiac RyR channels were found to exhibit a relatively high level of basal phosphorylation at S2809 (76.5 ± 9.8 % of maximum (SD; n=3)) that was associated with relatively low P_o under various conditions (0.050 ± 0.019 in the presence of 10 μM Ca²⁺). Despite this high level of phosphorylation, addition of the catalytic subunit of PKA to the cytosolic channel face, in the presence of ATP and Mg²⁺, resulted in a significant increase in P_o that was maintained after PKA was removed (0.145 ± 0.077 to 0.643 ± 0.197 (p<0.05) in the presence of 10 μM Ca²⁺, 10 mM ATP, and 0.5 mM Mg²⁺). The increase in P_o was also associated with a significant increase in single-channel current amplitude at a holding potential of 0 mV (4.12 ± 0.09 to 4.53 ± 0.05 pA and 3.69 ± 0.16 to 4.12 ± 0.16 pA (p>0.01) in the presence of 50 μM Ca²⁺ and 10 μM Ca²⁺, 10 mM ATP, and 0.5 mM Mg²⁺ respectively). In contrast, maximum PKA-dependent phosphorylation at S2809, as demonstrated by phosphorylation state specific antibodies (3), led to a significant reduction in [³H]ryanodine binding to 78.1 ± 2.1%, 77.7 ± 0.4%, and 29.8 ± 5.7% (p<0.01) of control binding at free [Ca²⁺] of 50 μM, 10 μM, and 1 μM. Our results indicate that increases in the level of phosphorylation at S2809 produce little change in channel function until very high or maximum levels of phosphorylation are reached, at which point, maintained high P_o values and increased conductance are achieved. Since our [³H]ryanodine binding studies suggest that phosphorylation of S2809 leads to a decrease in P_o, we conclude that the overall effect of phosphorylation at S2809 may depend on a complex combination of factors influencing channel gating including the presence of other associated proteins.