We undertook these studies to explore the intracellular signaling mechanisms activated by a newly described human brain melanocortin receptor (hMC3R). Hepa cells transfected with the hMC3R gene responded to stimulation with alpha-melanocyte stimulation hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) with dose-dependent increases in cellular content of cyclic 3',5'-adenosine monophosphate (cAMP) reaching a maximum of over 1500% of control cells at the 10(-8) M dose (EC50 = 10(-11) M). In contrast, the production of [3H]inositol phosphates in cells prelabeled with myo-[2-3H]inositol exhibited a biphasic dose-response curve with increases as high as 155% of basal at 10(-11) M alpha-MSH or ACTH, but beyond that a dose-dependent decrease was observed. The inhibitory component of the dose-response curve could be abolished by pretreatment of transfected cells with the cAMP antagonist (Rp)-adenosine 3',5'-monophosphorothioate (Rp-cAMP) or the protein kinase A inhibitor H-89. Increases in intracellular calcium induced in transfected cells by alpha-MSH in doses ranging from 10(-11) to 10(-7) M could not be observed unless the cells were pretreated with H-89. By replacing the third intracytoplasmic loop of the canine H2-histamine receptor with that of hMC3R the biphasic characteristic of agonist-induced production of [3H]inositol phosphates was conferred to the chimeric receptor. These data indicate that the hMC3R is coupled to both cAMP and inositol phospholipid/Ca(2+)-mediated post-receptor signaling systems and that the latter response is regulated by protein kinase A activity.