Using a specific antiserum raised against synthetic neuropeptide Y (NPY), the distribution of immunoreactivity in the brain and pituitary of the elasmobranch fish Scyliorhinus canicula has been examined with the indirect fluorescence and the peroxidase-antiperoxidase methods. The highest density of NPY-immunoreactive neurons was found in the basal telencephalon and in the hypothalamus. Numerous NPY-containing perikarya were located in the entopeduncular and the preoptic nuclei, in the nucleus lobi lateralis and in the nucleus lateralis tuberis. NPY-immunopositive fibers were observed throughout the fish brain. In particular, dense networks of fibers were present in the entopeduncular and the habenular nuclei, in the nucleus tuberculi posterioris and in the lateral lobes. Scattered fibers were observed in all other parts of the brain except in the cerebellum where no NPY-immunoreactive material could be detected. A plexus of NPY-immunoreactive fibers arising from the preoptic neurosecretory complex appeared to run through the basal hypothalamus and the pituitary stalk. These fibers terminated in the intermediate lobe of the pituitary, suggesting that NPY may be involved in the control of melanotropin secretion. The NPY-immunoreactive material localized in the brain and pituitary was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. Brain and pituitary extracts showed a good cross-reactivity to the NPY antiserum, but serial dilutions of tissue samples did not completely parallel the standard curve. HPLC analysis resolved two major forms of immunoreactive NPY in the hypothalamus while the pars intermedia contained only authentic NPY. The widespread distribution of NPY neurons in the fish brain and pituitary suggests the involvement of NPY in a variety of physiological functions, including the neuroendocrine control of the pituitary.