We have previously reported that neuropeptide-Y (NPY) stimulates GH and gonadotropin-II (GtH-II) release from perifused pituitary fragments in the goldfish. Since the teleost pituitary is directly innervated by neurosecretory terminals from the brain, we further investigated the possible sites of action of NPY. Both synthetic human NPY and NPY-(18-36), an agonist selective for the NPY Y2-receptor, stimulated GH and GtH-II release from the pituitary fragments; the magnitude of the response to NPY (18-36) was smaller than that to the whole molecule of NPY. NPY also stimulated the release of GH and GtH-II from perifused dispersed pituitary cells. In contrast, NPY-(18-36) had no effect on either GH or GtH-II release from dispersed pituitary cells. These data suggest that Y2 action is not direct at the level of pituitary cells, but may be indirect through actions on nerve terminals in the pituitary. The hypothesis that the action of NPY on GH and GtH-II release is mediated in part by GnRH was then tested. Both NPY and NPY-(18-36) stimulated the GnRH release from preoptic-anterior hypothalamic slices and pituitary fragments with similar potency. Furthermore, a GnRH antagonist significantly reduced the effects of NPY on both GH and GtH-II release in perifused pituitary fragments. Similar to previous findings, NPY, when given at 55-min intervals, desensitized the hormone responses in pituitary fragments. Similarly, the same treatment with NPY in perifused dispersed pituitary cells induced desensitization of GH and GtH-II responses. Together, these results suggest that 1) more than one type of NPY receptors are present in the goldfish pituitary; and 2) NPY has at least two sites of action in the pituitary. One site of action is the pituitary cells, where NPY directly stimulates GH and GtH-II secretion; the second is the nerve terminals, where NPY presynaptically stimulates GnRH release via Y2-like receptors, and GnRH, in turn, stimulates GH and GtH-II release.