To investigate the functional relevance of the G-protein G(o) to adenosine-induced effects in the heart, we studied the localization of G(o) and its interaction with A1 adenosine receptors. Concentrations of G(o) in various mammalian hearts differed markedly between the atrial and the ventricular muscle, as well as among species. In most species examined, the concentration of G(o) was much higher in the atrium than in the ventricle. The highest levels of G(o) in atria and ventricles were found in the ferret heart. An immunohistochemical study of the ferret heart with G(o) alpha-specific antibodies showed that G(o) was localized throughout the membranes of cardiac myocytes, including the intercalated disks. In addition, G(o) was densely distributed in the nerve fibers and Purkinje fibers. Analyses of G(o) alpha subtypes showed that bovine atrium mainly contained G(o)A alpha, while bovine ventricle contained only G(o)B alpha. By contrast, ferret ventricle contained both subtypes of G(o) alpha. To study the coupling of G(o) to A1 adenosine receptors, receptors in ventricular membranes of ferrets, which had been pretreated with pertussis toxin, or purified receptors were reconstituted with purified G(o)A and G(o)B. The reconstitution experiments indicated that both subtypes of G(o) coupled with A1 adenosine receptors. These results suggest that the effect of adenosine is mediated by both G(o)A and G(o)B in membranes of ferret cardiac myocytes.