We have already reported that A3 adenosine receptor stimulation reduces [3H]-ryanodine binding and sarcoplasmic reticulum Ca2+ release in rat heart. In the present work we have investigated the transduction pathway responsible for this effect. Isolated rat hearts were perfused for 20 min in the presence of the following substances: 100 nM N6-(iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA), an A3 adenosine agonist; 10 μM U-73122, a phospholipase C inhibitor; 2 μM chelerythrine, a protein kinase C inhibitor. At the end of perfusion, the hearts were homogenized and [3H]-ryanodine binding was assayed. IB-MECA produced a significant decrease in ryanodine binding, which was abolished in the presence of chelerythrine but not in the presence of U-73122. RT-PCR experiments showed that ryanodine receptor gene expression was not affected by IB-MECA. In Western blot experiments, ryanodine receptor phosphorylation on serine 2809 was not modified after perfusion with IB-MECA. We conclude that modulation of SR Ca2+ release channel by IB-MECA is dependent on protein kinase C activation. However, in this model protein kinase C activation is not due to phospholipase C activation. In addition, changes in ryanodine receptor gene expression or direct phosphorylation of the ryanodine receptor on serine 2809 residue do not appear to occur.