Abstract--Adenosine acts as an important protector of ischemic myocardium through coronary vasodilation and the depression of cardiac contractility. The protective effect of adenosine may partly relate to the cardiac hormone atrial natriuretic peptide (ANP). The aim of the present study was to investigate the effects of adenosine and the adenosine receptor subtype on atrial hemodynamics and ANP release using isolated perfused beating rat atria. Adenosine, a nonselective adenosine receptor agonist, increased the ANP release with negative inotropism in a dose-dependent manner. Adenosine-stimulated ANP release was attenuated by a selective A₁ antagonist but not A_2A antagonist or A₃ antagonist. The order of potency of the various agonists for the ANP release was A₁ agonists>>A₃ agonist=adenosine>A_2A agonist. The order of potency for the negative inotropy was A₁ agonists>adenosine=A_2A agonist>A₃ agonist. The negative inotropism and ANP release by a specific A₁ agonist (N⁶-cyclopentyl-adenosine) were also attenuated by A₁ antagonist but not A_2A antagonist or A₃ antagonist. Treatment with A₁ agonist resulted in a decrease of cAMP contents in atria and perfusates. The agonist-stimulated ANP release was significantly attenuated in the presence of forskolin, isoproterenol 8-Br-cAMP, or an adenylyl cyclase inhibitor. These results suggest that the A₁ receptor subtype is responsible for the adenosine-induced ANP release and negative inotropism through adenylyl cyclase-cAMP pathway.