Although there is considerable interest in the regulation of the different beta-adrenergic receptor (AR) subtypes, most previous studies have utilized stably transfected cells expressing recombinant receptors under the control of viral promoters. Human SK-N-MC neurotumor cells appear to be novel, since they express both endogenous beta 1AR and beta 3AR based on radioligand binding and on functional response. Saturation binding of either the hydrophilic ligand (-)-[3H]CGP-12177 or the more hydrophobic (-)-[125I]iodocyanopindolol indicated the presence of two populations of binding sites with high and low affinities. With either ligand, the beta 1AR antagonist CGP-20712A preferentially inhibited binding to the high-affinity sites. This is consistent with the latter representing beta 1AR whereas the low-affinity sites represent beta 3AR. Both subtypes appeared to be functional on the basis of isoproterenol stimulation of cyclic adenosine monophosphate (cAMP) in intact cells and adenylyl cyclase activity in cell membranes in the absence and presence of CGP-20712A. SK-N-MC-IXC cells, derived by twice subcloning the parental cells, also expressed both beta AR subtypes, indicating that they co-exist in the same cell. SK-N-MC cells exposed to isoproterenol exhibited a rapid sequestration and a slower downregulation of beta 1AR. The latter subtype also underwent desensitization, as indicated by a rightward shift to less sensitivity in the EC50 for isoproterenol stimulation of adenylyl cyclase activity. In contrast, the beta 3AR subtype was resistant to agonist-mediated sequestration, downregulation, and desensitization. Thus, when endogenously expressed in the same cell line, human beta 1AR and beta 3AR display differences in their ability to be regulated by agonist.