Multiple forms of urotensin II (UII), one of the hormonal peptides of the caudal neurosecretory system of fishes, were purified from the urophyses of the carp, Cyprinus carpio. Three distinct peaks with UII activity (classified as UII-alpha, -beta and -gamma) were separated by reverse-phase high-pressure liquid chromatography (HPLC). Edman degradation as well as digestion with carboxypeptidase A revealed the primary structures of these peptides as UII-alpha: Gly-Gly-Gly-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val UII-beta: Gly-Gly-Ser-Asn-Thr-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val UII-gamma: Gly-Gly-Gly-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Ile The results of thin-layer chromatography, HPLC, amino acid analysis, and sequencing indicate that UII-alpha and -gamma are homogeneous. UII-beta appears, however, to be a mixture of two components, differing only at position 2. Thus, in the carp urophysis, four forms of UII appear to be present, although the separation of two components in UII-beta has not been obtained. Sequence of positions 6-11 is common to all forms of UII isolated from the carp, sucker (Catostomus commersoni) and goby (Gillichthys mirabilis).