The minimal sequence required for biological activity of alpha-MSH (alpha-melanotropin, alpha-melanocyte stimulating hormone) was determined in the frog (Rana pipiens) skin bioassay. The sequence required to elicit measurable biological activity was the central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2 (Ac-alpha-MSH6-9-NH2), which was about 6 orders of magnitude less potent than the native tridecapeptide. Smaller fragments of this sequence (Ac-His-Phe-NH2, Ac-Phe-Arg-NH2, Ac-His-Phe-Arg-NH2) were devoid of melanotropic activity at concentrations as high as 10(-4) M. We were unable to demonstrate biological activity for the tetrapeptide, Ac-Phe-Arg-Trp-Gly-NH2 (Ac-alpha-MSH7-10-NH2), and for several carboxy terminal analogues including Ac-Lys-Pro-Val-NH2 (Ac-alpha-MSH11-13-NH2). We prepared a series of fragment analogues of alpha-MSH in an attempt to determine the contribution of each individual amino acid to the biological activity of the native hormone. The minimal potency of Ac-alpha-MSH6-9-NH2 could be enhanced about a factor of 16 by the addition of glycine to the C-terminus, yielding Ac-alpha-MSH6-10-NH2 (Ac-His-Phe-Arg-Trp-Gly-NH2). Addition of glutamic acid to the N-terminus provided the peptide, Ac-alpha-MSH5-10-NH2, which was only slightly more potent than Ac-alpha-MSH6-10-NH2, indicating that position 5 contributes little to the biological potency of alpha-MSH in this assay. Addition of methionine to the N-terminus of Ac-alpha-MSH5-10-NH2 resulted in the heptapeptide, Ac-alpha-MSH4-10-NH2, which was only about 4-fold more potent than Ac-alpha-MSH5-10-NH2. Addition of lysine and proline to the C-terminal of the Ac-alpha-MSH4-10-NH2 sequence yielded the peptide, Ac-alpha-MSH4-12-NH2 with a 360-fold increase in potency relative to Ac-alpha-MSH4-10-NH2. This peptide was only about 6-fold less potent than alpha-MSH. A series of Nle-4-substituted analogues also were prepared. Ac-[Nle4]-alpha-MSH4-10-NH2 was about 4 times more potent than Ac-alpha-MSH4-10-NH2. Ac-[Nle4]-alpha-MSH4-11-NH2 also was about 4 times more potent than Ac-alpha-MSH4-10-NH2, demonstrating that lysine-11 contributes somewhat to the biological activity of alpha-MSH on the frog skin melanocyte receptor.(ABSTRACT TRUNCATED AT 250 WORDS)