Neuropeptide Y was isolated from the brain of the Atlantic cod, Gadus morhua and its primary structure established as Tyr-Pro-Ile*-Lys-Pro-Glu*-Asn-Pro-Gly-Glu10-Asp-Ala-Pro-Ala-Ala-Asp*-Glu*-Leu*-Ala-Lys*-Tyr20-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Ile-Thr-Arg-Gln-Arg-Tyr-CONH2. Residues denoted by an asterisk are different from the corresponding sequence of human neuropeptide Y. A structurally similar peptide was isolated from the brain of the trout, Oncorhynchus mykiss. Trout neuropeptide Y contains four substitutions (Ile3→Val, Ala14→Thr, Asp15→Glu and Ser22→Thr) compared with cod neuropeptide Y. A second peptide of the neuropeptide Y family was identified in the trout brain and this component was structurally similar to peptide tyrosine-tyrosine previously isolated from frog intestine (six amino acid substitutions) and identical to a peptide isolated from the pancreas of the closely related species, Oncorhynchus kisutch (Coho salmon). Peptide tyrosine-tyrosine, with the same primary structure as the brain peptide, was also isolated from an extract of the trout stomach. The data indicate that a peptide analogous to mammalian neuropeptide Y is present in the brain of teleost fish and a peptide analogous to mammalian peptide tyrosine-tyrosine is present in brain, gastrointestinal tissue and pancreas. We speculate, therefore, that the putative gene duplication that led to pancreatic polypeptide in the higher vertebrates took place after the time of divergence of fish and tetrapods.