A Trp-->Arg mutation at amino acid position 64 in the human beta3-adrenoceptor is reportedly associated with morbid obesity; carriers suffer from increased gain in mass, early-onset diabetes, insulin resistance, and an increased waist-to-hip ratio [Clément, K., Vaisse, C., Manning, B. S., Basdevant, A., Guy-Grand, B., Ruiz, J., Silver, K. D., Shuldiner, A. R., Froguel, P. & Strosberg, A. D. (1995) N. Engl. J. Med. 333, 352-354]. Here, we report the stable expression of the genes encoding the wild-type or the [Arg64]beta3-adrenoceptor in two different cell types: hamster CHO-K1 and human HEK293. The mutated receptor displayed unchanged pharmacological values compared to the wild type for the binding inhibition (Ki) and adenylyl cyclase activation constants (K(act)) in two independent clones of both cell lines. However, maximal cAMP accumulation was significantly reduced in response to various beta3-adrenergic agonists, including endogenous catecholamines, (-)-epinephrine and (-)-norepinephrine, the non-selective agonist (-)-isoproterenol, and the beta3-adrenergic selective agonist CGP 12177A. Treatment with Pertussis toxin did not restore the adenylyl cyclase activity to that of the wild type, suggesting that the reduction in cAMP accumulation observed in cells expressing [Arg64]beta3-adrenoceptor is not due to enhanced interaction of the beta3-adrenoceptor with the inhibitory Gi protein.