Twenty nine analogs of a superpotent MC1R agonist LK-184 (1) were tested at human melanocortin receptors (hMC1, hMC3, and hMC4Rs). All derivatives with the spacer between the N-terminus and the aromatic ring longer or shorter than C(3) were much less potent at hMC1R than 1. Only LK-312 PhCO(CH(2))(3)CO-His-d-Phe-Arg-Trp-NH(2) (3), partially mimicking the pi-system of 1, had an EC(50) of 0.05 nM at hMC1R, which confirms the localization of the pi-binding zone of the receptor. Truncation of 1 to Ph(CH(2))(3)CO-His-d-Phe-Arg-NH(2) gave a full MC1 agonist, LK-394 (30), with an EC(50) of 5 nM and a weak partial agonism at MC3/4Rs. This suggests the existence of an additional binding site within hMC1R next to that for the core sequence His-d-Phe-Arg-Trp-NH(2).