Adenosine receptors are widely distributed in mammalian tissues and have been possibly involved through transmembrane potential changes in cell function regulation. The effect of A₁ and A_2A adenosine receptor ligands on transmembrane potential measured with flow cytometry and potassium conductance measured by the patch-clamp technique was investigated in DDT1 MF-2 smooth muscle cells. The A₁ adenosine-receptor agonist CPA (50 nM) and the A2A adenosine-receptor agonist CGS 21680 (50 nM) elicited a rapid and maintained increase and decrease in the potassium conductance, respectively, and a concomitant hyperpolarization and depolarization of the membrane, respectively. These effects were eliminated by subtype-selective adenosine receptor antagonists (DPCPX, CSC, ZM 241385, all 1 μ M). The ligand induced membrane potential changes were reversible. Based on these detected membrane potential changes along with the published voltage dependence of the adenylyl cyclase, the regulation of cAMP production by A₁- and A_2A-receptor activation is suggested to be mediated through the induced early hyperpolarization and depolarization. The interaction between the effects of these receptor subtypes allows for a complex regulation mechanism.