Adenosine is a potent mediator of myocardial protection against hypertrophy via A1 or A3 receptors that may be partly related to atrial natriuretic peptide (ANP) release. However, little is known about the possible involvement of the A3 receptor on ANP release. We studied the effects of the A3 receptor on atrial functions and its modification in hypertrophied atria. A selective A3 receptor agonist, 2-chloro-N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (2-CI-IB-MECA), was perfused into isolated, beating rat atria with and without receptor modifiers. 2-CI-IB-MECA dose-dependently increased the ANP secretion, which was blocked by the A3 receptor antagonist, but the increased atrial contractility and decreased cAMP levels induced by 30 μM 2-CI-IB-MECA were not affected. The 100 μM 2-(1-hexylnyl)-N-methyladenosine (HEMADO) and N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA), A3 receptor agonist, also stimulated the ANP secretion without positive inotropy. The potency for the stimulation of ANP secretion was 2-CI-IB-MECA >> IB-MECA = HEMADO. The inhibition of the ryanodine receptor or calcium/calmodulin-dependent kinase II (CaMKII) attenuated 2-CI-IB-MECA-induced ANP release, positive inotropy, and translocation of extracellular fluid. However, the inhibition of L-type Ca2+ channels, sarcoplasmic reticulum Ca2+-reuptake, phospholipase C or inositol 1,4,5-triphosphate receptors did not affect these parameters. 2-CI-IB-MECA decreased cAMP level, which was blocked only with an inhibitor of CaMKII or adenylyl cyclase. These results suggest that 2-CI-IB-MECA increases the ANP secretion mainly via A3 receptor activation and positive inotropy by intracellular Ca2+ regulation via the ryanodine receptor and CaMKII.