Adenosine A1-receptor-mediated inhibition of exocytotic norepinephrine (NE) release from sympathetic nerve endings has been implicated as an endogenous cardioprotective mechanism. So far, the intraneuronal signal transduction underlying the adenosine A1-receptor-elicited inhibition of NE release is not known. In the present study, we determined in isolated Langendorff-perfused rat hearts the role of inhibitory G-proteins and of adenylyl cyclase (AC) on NE release after pharmacologic adenosine A1-receptor activation. NE release was induced by electrical field stimulation and was assessed in the coronary effluent by high-performance liquid chromatography. Adenosine A1-receptor activation with 2-chloro-N6-cyclopentyladenosine (CCPA) decreased NE release by approximately 50% in hearts from both untreated and pertussis toxin-pretreated rats. In hearts from untreated rats, suppression of NE release in response to CCPA was completely abolished by the cell-permeable AC inhibitor 9-(tetrahydro-2'-furyl)adenine (SQ 22536). Direct activation of AC with forskolin increased NE release by approximately 20%. In the presence of forskolin, stimulation of adenosine A1-receptors with CCPA or inhibition of AC with SQ 22536 decreased NE release to baseline. These findings suggest a Gi-protein-independent but AC-dependent inhibition of NE release following adenosine A1-receptor activation.