CRH and urotensin I (UI) are neuroendocrine peptides that belong to the superfamily of corticotropin-releasing factors. In mammals, these peptides regulate the stress response and other central nervous system functions, whereas in fish an involvement for UI in osmoregulation has also been suggested. We have identified, characterized, and localized the genes encoding these peptides in a unique fish neuroendocrine organ, the caudal neurosecretory system (CNSS). The CRH and UI precursors, isolated from a European flounder CNSS library, consist of 168 and 147 amino acid residues, respectively, with an overall homology of approximately 50%. Both precursors contain a signal peptide, a divergent cryptic region and a 41-amino acid mature peptide with cleavage and amidation sites. Genomic organization showed that whole CRH and UI coding sequences are contained in a single exon. Northern blot analysis and quantitative PCR of a range of tissues confirmed the CNSS as a major site of expression of both CRH and UI and thus serves as a likely source of circulating peptides. In situ hybridization demonstrated that CRH and UI colocalize to the same cells of the CNSS. Our findings suggest that, in euryhaline fish, the CNSS is a major site of production of CRH and probably contributes to the high circulating levels observed in response to specific environmental challenges. Furthermore, the localization of CRH and UI within the same cell population suggests an early, possibly shared role for these peptides in controlling stress-mediated adaptive plasticity.