One of the most successful chromatic adaptations in vertebrates is the dorsal-ventral (D-V) pigment pattern in which the dorsal skin is darkly colored while the ventrum is light. In fish, the latter pattern is achieved because a melanization inhibition factor (MIF) inhibits melanoblast differentiation and supports iridophore proliferation in the ventrum. In rodents, the patterned pigmentation results from regional production of the agouti-signaling protein (ASP). This peptide controls the switch between production of eumelanin and phaeomelanin by antagonizing -melanocyte stimulating hormone (-MSH) effects on melanocortin receptor 1 (MC1R) in the melanocytes. In addition, ASP inhibits the differentiation and proliferation of melanoblast. Thus, the mammalian ASP may be homologous to the poikilotherm MIF. By screening of a genomic library, we deduced the amino acid sequence of goldfish ASP. The ASP gene is a four-exon gene spanning 3097 bp that encodes a 125-amino acid precursor. Northern blot analysis identified two different ASP mRNAs in ventral skin of red-, black-pigmented and albino fish but no expression levels were observed in the dorsal skin of the same fish. The dorsal-ventral expression polarity was also detected in both black-dorsally pigmented fish and albino fish. Pharmacological studies demonstrate that goldfish ASP acts as a melanocortin antagonist at Fugu MC1R and goldfish MC4R. In addition, goldfish ASP inhibited NDP-MSH-stimulated pigment dispersion in medaka melanophores. Our studies support agouti signaling protein as the melanization inhibition factor, a key factor in the development of the dorsal-ventral pigment pattern in fish.