A pigment-dispersing hormone (PDH) from eyestalks of the fiddler crab Uca pugilator has been purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase liquid chromatography. Based on automated gas-phase sequencing and subsequent identification of carboxyl-terminal amide, we have assigned the primary structure of this peptide as Asn-Ser-Glu-Leu-Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Asp-Ala-NH (2). We have confirmed the sequence by synthesizing this peptide and demonstrating that the synthetic PDH and the native PDH display identical chromatographic behavior and biological activity. This hormone is a member of a family of invertebrate neuropeptides that includes a light-adapting/pigment-dispersing octadecapeptide hormone from the prawn Pandalus borealis. In assays for melanophore pigment dispersion in destalked fiddler crabs, Uca PDH was 21-fold more potent than Pandalus PDH. These two hormones share a hexapeptide core sequence (residues 5-10: -Ile-Asn-Ser-Ile-Leu-Gly-) as well as the amino- and carboxyl-terminal residues but differ at positions 3, 4, 11, 13, 16, and 17. These results point to speciesrelated or group-specific structural differences among crustacean PDHs.