Previous studies established that beta 2-adrenergic receptors enhance the amplitude, without abbreviating the kinetics, of the twitch in adult rat ventricular myocytes. The present study was designed to identify the dominant signaling mechanism mediating this response. Myocytes from adult rat ventricles were loaded with the pH-sensitive fluorophore 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein, and simultaneous measurements of intracellular pH and contraction were performed during electrical field stimulation under basal conditions and after stimulation with isoproterenol or the selective beta 2-receptor agonist zinterol. Inhibition of protein kinase A with H-7 completely inhibited the isoproterenol-dependent, but not the zinterol-dependent, positive inotropic response. The effect of zinterol to increase twitch amplitude was associated with an alkalinization of 0.07 +/- 0.02 pH unit, which was not prevented by inhibition of the Na+/H+ exchanger with hexamethylene-amiloride. Rather, removal of bicarbonate from the extracellular buffer prevented the beta 2-receptor-dependent alkalinization as well as the positive inotropic response. These results indicate that beta 2-adrenergic receptors induce a positive inotropic response in adult rat ventricular myocytes via a adenosine 3',5'-cyclic monophosphate-independent mechanism that involves intracellular alkalinization due to activation of a bicarbonate-dependent intracellular pH regulatory mechanism.