To further evaluate elements that could contribute to the 3D topographical structure of gamma-MSH, we have systematically designed a group of linear gamma-MSH analogues and evaluated their biological activities: without a N-terminal acetyl, with and without a C-terminal amide, with Nle(3), with l- or d-Phe(6) or d-Nal(2')(6), and with d-Trp(8) or d-Nal(2')(8). It was found that changing the C-terminal acid in gamma-MSH to an amide and replacing Met with Nle leads to increased binding affinities at all four subtypes of melanocortin receptors (10-100 fold). Substitution of Trp(8) with d-Nal(2')(8) and Phe(6) with d-Phe(6) in gamma-MSH-NH(2) forms a selective antagonist for the hMC3R, whereas, substitution of Phe(6) with d-Nal(2')(6) and replacing Trp(8) with d-Trp(8) at gamma-MSH-NH(2) yields a selective partial agonist for the hMC1R. Finally, substitution of His(5) with Pro(5) and Trp(8) with d-Nal(2')(8) in gamma-MSH-NH(2) leads to a highly potent and selective agonist for the hMC1R. Molecular modeling showed that, at the C-terminal of Nle(3)-gamma-MSH-NH(2), there is a reverse-turn-like structure, suggesting that there might be a secondary binding site involved in ligand-receptor interaction for gamma-MSH analogues that may explain the enhanced binding affinities of the Nle(3)-gamma-MSH-NH(2) analogues. Our results indicate that increasing the hydrophobicity and replacing Phe(6) and Trp(8) with bulkier aromatic amino acid residues is very important for selectivity of alpha-MSH/gamma-MSH hybrids for hMCRs.