Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) is a class A G protein-coupled receptor (GPCR). The MCH system has been linked to a variety of physiological functions, including the regulation of feeding and energy metabolism. We recently reported the importance of a dibasic motif in the membrane-proximal C-terminal region for MCH1R function. Here, we reveal that an Arg residue in intracellular loop 2 of MCH1R plays a critical role in receptor function. We analyzed the roles of two distinct motifs, BBXXB and BXBB (where B is a basic residue and X is a nonbasic residue), located in the three intracellular loops of MCH1R. Triple-substitution mutants of intracellular loops 1 and 3 could still activate calcium mobilization, albeit with lower efficacy or potency. However, mutations in intracellular loop 2 led to a complete loss of induction of signal transduction without changing the high-affinity Kd value. By analyzing a series of single-substitution mutants, a point mutation of Arg (155) in intracellular loop 2 was found to be responsible for the signaling pathway elicited by MCH. In addition, substitution at positions corresponding to Arg (155) in human MCH receptor 2 and rat somatostatin receptor 2 also markedly abolished their ligand-induced signaling capacities, indicating that this Arg is a recognition determinant in several GPCRs.