Glycogenolysis in hepatocytes isolated from fed guinea pigs was much more enhanced by the beta-agonist, isoproterenol, than by equimolar concentrations of the alpha-agonists, phenylephrine and norepinephrine. The stimulatory effects of catecholamines occurred with the following order of potency: isoproterenol greater than epinephrine greater than norepinephrine. This order of potency is characteristic of beta 2-adrenergic receptors. That beta 2-receptors are responsible for mediating catecholamine stimulation of glycogenolysis in guinea-pig hepatocytes was further deduced from the inhibition of agonist-stimulated glycogenolysis by beta-receptor sub-type-selective antagonists. Thus, IPS 339, a beta-antagonist which has higher affinity at beta 2-sites than at beta 1-sites, was three orders of magnitude more potent in inhibiting isoproterenol-stimulated glycogenolysis than either atenolol or practolol, both of which are beta 1-selective antagonists. The beta 2-agonists zinterol and procaterol also stimulated glycogenolysis in hepatocytes and their effects were inhibited by propranolol and IPS 339, but not by practolol. Furthermore, activation of phosphorylase in these hepatocytes by isoproterenol, epinephrine, and norepinephrine also occurred with the potency order expected for beta 2-receptors. These results are in sharp contrast to those obtained with rat hepatocytes and emphasize that species differences occur in the regulation of hepatic glycogenolysis by catecholamines.