Since the melanocortin MC3 and melanocortin MC4 receptors are the main melanocortin receptor subtypes expressed in rat brain, we characterized the activity and affinity of nine melanocortin receptor ligands using these receptors in vitro, as well as their activity in a well-defined melanocortin-induced behavior in the rat: grooming behavior. We report here that [D-Tyr4]melanotan-II and RMI-2001 (Ac-cyclo-[Cys4, Gly5, D-Phe7, Cys10]alpha-MSH-NH2) have significantly higher affinity and potency on the rat melanocortin MC4 receptor as compared to the rat melanocortin MC3 receptor. Nle-gamma-MSH (melanocyte-stimulating hormone) was the only ligand with higher affinity and potency on the rat melanocortin MC3 receptor. The potency order of melanocortin MC4 receptor agonists, but not that of melanocortin MC3 receptor agonists, fitted with the potency of these ligands to stimulate grooming behavior, when administered intracerebroventricularly. SHU9119 (Ac-cyclo-[Nle4, Asp5, D-Nal(2)7, Lys10]alpha-MSH-(4-10)-NH2) and RMI-2005 (Ac-cyclo-[Cys4, Gly5, D-Na](2)7, Nal(2)9, Cys10]alpha-MSH-(4-10)-NH2) were able to inhibit alpha-MSH-induced melanocortin receptor activity in vitro, as well as alpha-MSH-induced grooming behavior. Melanotan-II, [Nle4-D-Phe7]alpha-MSH and RMI-2001 were also effective in inducing grooming behavior when administered intravenously. In the absence of purely selective melanocortin MC(3/4) receptor ligands, we demonstrated that careful comparison of ligand potencies in vitro with ligand potencies in vivo, could identify which melanocortin receptor subtype mediated alpha-MSH-induced grooming behavior. Furthermore, blockade of novelty-induced grooming behavior by SHU9119 demonstrated that this physiological stress response is mediated via activation of the melanocortin system.