The role of P₁ purinoceptor subtypes, the adenylyl cyclase (AC) pathway, and ATP-sensitive K⁺ (K_ATP) channels in adenosine (Ado)-induced membrane hyperpolarization was investigated in isolated segments of the guinea pig coronary artery using conventional microelectrode techniques. Ado (1-100 µM) elicited concentration-dependent hyperpolarization (half-maximal effective concentration 7.5 ± 0.5 µM) that averaged 28.6 ± 2.9 mV at 100 µM Ado. The A₁ selective agonist N⁶-cyclopentyladenosine (CPA), the A₁/A₂ agonist 2-chloroadenosine, and the A_2a/A_2b agonist 5-(N-ethylcarboxamido)adenosine (NECA) each induced glibenclamide (3 µM)-sensitive hyperpolarization at 10 µM. However, the selective A_2a-receptor agonists CGS-21680 and N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine (10 µM each) were without effect. Responses to CPA and NECA were significantly reduced by the AC inhibitor SQ-22,536 (100 µM). Activation of the AC-adenosine 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway by four additional methods, i.e., 1) forskolin (0.3-1 µM), 2) isoproterenol (0.1-1 µM), 3) combined milrinone (0.4 µM) and rolipram (30 µM), and 4) combined N⁶-phenyladenosine 3',5'-monophosphate, 8-(6-aminohexyl)aminoadenosine 3',5'-cyclic monophosphate, and the Sp-isomer of 5,6-dichloro-1-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphothioate (100 µM each), also gave rise to glibenclamide-sensitive hyperpolarization. These results suggest that stimulation of A_2b receptors coupled to AC represents the predominant mechanism by which Ado elicits hyperpolarization in this vessel. The ensuing increase in cAMP activates PKA, which then increases the activity of K_ATP channels. Our results further suggest that K_ATP channels are an important target for numerous pathways that raise cAMP levels in the coronary artery.