The ACTH receptor [melanocortin-2 receptor (MC2R)] is the smallest known G protein-coupled receptor (GPCR). Herein, human MC2R accessory protein (MRAP) isoforms alpha and beta, cloned from a human fetal adrenal gland, were expressed with c-Myc-tagged MC2R (Myc-MC2R) in 293/Flp recombinase target site cells by homologous recombination. Although insertion of Myc-MC2R at the plasma membrane occurred without MRAP assistance, ACTH stimulation of cAMP production was only detected in cells coexpressing MC2R with either MRAP isoform. On the other hand, a MC2R-green fluorescent protein fusion transfected with either MRAPalpha or MRAPbeta was impaired both in cell membrane localization and signaling. MRAP isoforms were also tagged with either Flag or 6xHis epitopes. In cell populations coexpressing transiently and/or stably Myc-MC2R with MRAPalpha or MRAPbeta, stimulation with ACTH induced production of cAMP with EC(50) values lower in MRAPalpha- than in MRAPbeta-expressing cells. ACTH only bound Myc-MC2R in the presence of MRAP. Higher Myc-MC2R cell surface density was observed in the presence of MRAPbeta comparatively to MRAPalpha, possibly contributing to higher ACTH binding capacity and higher maximal cAMP responses observed in MRAPbeta-expressing cells. Immunofluorescence studies indicated that MRAP isoforms were localized near the plasma membrane and in the vicinity, but not colocalized, with Myc-MC2R. In summary, through the generation of a new all-human experimental model devoid of endogenous MCRs, we present evidence that human MRAP isoforms, although not essential for MC2R localization at the plasma membrane, are essential for ACTH binding and ACTH-induced cAMP production and that they differentially regulate, although modestly, cell membrane density and functional properties of MC2R.