Utilizing results from previous structure-activity relationships and theoretical studies of alpha-melanotropin (alpha-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and its related superpotent analogues, Ac-[Nle4,D-Phe7]-alpha-MSH and Ac-[Cys4,Cys10]-alpha-MSH, we have designed a new class of alpha-MSH4-13 and alpha-MSH4-10 cyclic lactam fragment analogues of alpha-melanotropin. The cyclic peptides have the following general structures: Ac-[Nle4,Xxx5,D-Phe7,Yyy10,Gly11]-alpha-MSH4-13- NH2 and Ac-[Nle4,Xxx5,D-Phe7,Yyy10]-alpha-MSH4-10-NH2, where Xxx = Glu or Asp and Yyy = Lys, Orn, Dab, or Dpr. Formation of the lactam bridge between the side-chain groups Xxx and Yyy was performed either in solution or on a solid-phase support. Seven cyclic peptides were prepared and bioassayed for their melanotropic potency by using standard frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. Relative to alpha-MSH (relative potency = 1), the potencies of the cyclic peptides in the lizard skin bioassay were as follows: alpha-MSH (1); Ac-[Nle4,Glu5,D-Phe7,Lys10,Gly11]-alpha-MSH4-13- NH2 (6); Ac-[Nle4,Asp5,D-Phe7,Lys10,Gly11]-alpha-MSH4-13- NH2 (100); Ac-[Nle4,Glu5,D-Phe7,Lys10]-alpha-MSH4-10-NH2 (9); Ac-[Nle4,Asp5,D-Phe7,Lys10]-alpha-MSH4-10-NH2 (90); Ac-[Nle4,Asp5,D-Phe7,Orn10]-alpha-MSH4-10-NH2 (20); Ac-[Nle4,Asp5,D-Phe7,Dab10]-alpha-MSH4-10-NH2 (5); Ac-[Nle4,Asp5,D-Phe7,Dpr10]-alpha-MSH4-10-NH2 (5). Similar results were obtained in the frog skin bioassay, but the analogues were much less potent. Cyclic melanotropins with 23-membered rings exhibited 100-fold higher melanotropic potency than alpha-MSH with selectivity for the lizard melanocyte receptors over the frog melanocyte receptors. Increasing or decreasing the ring size of these cyclic melanotropins from 23 diminishes the biological potency of the resulting cyclic peptide. The 23- and 24-membered ring analogues showed prolonged (residual) biological activities in both biological assays, but the smaller ring systems (20, 21, 22) did not. These results provide new insights into the structural and conformational requirements of alpha-MSH and its analogues at two different types of pigment cell (melanocyte) receptors.