Secretoneurin (SN) is a 33-34 amino acid neuropeptide derived by endoproteolysis of secretogranin-II (SgII), a chromogranin. A multi-antigenic strategy was used to generate a rabbit polyclonal goldfish SN antiserum that was characterized for Western blot analysis. In the goldfish pituitary two intermediate proteins containing SN and likely processed from the 69.6-kDa SgII precursor were detected. No immunoreactive proteins were observed in the goldfish interrenal, ovary, cerebellum, and telencephalon whereas SgII mRNA was expressed in all these tissues. Immunoreactive levels of the approximately 57 kDa product were higher in the pars distalis (PD) than in the neurointermediate lobe (NIL). The abundance of the approximately 57 kDa protein indicates that this SgII-product containing the SN sequence is a major stored form in secretory granules of the goldfish pituitary. High expression and processing of SN in the hypothalamus and pituitary suggest important roles for SgII-derived peptides in neuroendocrine tissues.