Previous studies have suggested that the beta 3 receptor fails to become desensitized following acute agonist exposure. We have determined whether this resistance to desensitization is an intrinsic property of the human beta 3 receptor by examining beta 3 receptor-stimulated adenylyl cyclase activity in several cell types. Prior exposure to ISO significantly decreased beta 3 receptor-stimulated adenylyl cyclase activity in SK-N-MC human neuroepithelioma cells, which natively express the beta 3 receptor. ISO pretreatment significantly desensitized the recombinant beta 3 receptor when stably expressed in 293 cells, but not when the receptor was expressed in Chinese hamster ovary cells. Mutant receptors lacking the second exon of the human beta 3 receptor also underwent agonist-induced desensitization when expressed in 293 cells. Additionally, the rat beta 3 receptor, which fails to desensitize in rat adipocytes, underwent agonist-induced desensitization when expressed in 293 cells. Pretreatment with CGP 12177A, a beta 3-selective agonist, also reduced beta 3-stimulated adenylyl cyclase activity in transfected 293 cells. In contrast, 8-Br-Cyc AMP did not desensitize the beta 3 receptor. Concanavalin A, an inhibitor of receptor sequestration failed to prevent ISO-induced desensitization of the beta 3 receptor. Furthermore, radioligand binding studies showed that ISO pretreatment did not cause a loss of beta 3 receptors from 293 cell membranes. The results of the present study indicate that beta 3 receptor desensitization is dependent upon the cellular background in which this receptor is expressed. Furthermore, the mechanism responsible for beta 3 receptor desensitization does not appear to involve sequestration, cyclic AMP-dependent phosphorylation or down-regulation of the receptor.