The melanocortin 2 (MC2) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC2 receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC2 receptor responsible for these characteristics, we created chimeras between MC2 and MC4 receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC2 but not MC4 receptors. Replacing the first transmembrane segment of the MC2 receptor with the corresponding region from the MC4 receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC4 receptor with the corresponding regions from the MC2 receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC2 receptors with MC4 sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-D-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC2 receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second through third transmembrane segments of the MC2 receptor as important for MRAP dependent-signal transduction and ligand specificity.