Neuropeptide Y (NPY) increases LH secretion in part by enhancing the release of LH in response to GnRH. The present studies examined whether NPY influences the binding of GnRH to its receptors and also assessed whether specific binding sites for NPY exist in rat anterior pituitary membranes. In concentrations from 66-200 nM, NPY dose-dependently enhanced the binding of a 125I-labeled GnRH agonist, [D-Ala6, des-Gly10]GnRH ethylamide (GnRHa; 30 pM) to anterior pituitary membranes of chronically ovariectomized rats; higher concentrations of NPY were ineffective. At 200 nM, NPY significantly increased the high affinity binding of the GnRHa to these membranes, without change in the apparent maximum binding capacity. Further, 200 nM NPY significantly increased the binding of [125I]GnRHa when this tracer was used in concentrations of less than 100 pM, but NPY did not increase the binding of higher concentrations of [125I]GnRHa. Peptide YY, a gastrointestinal peptide structurally and functionally related to NPY, and the hypothalamic/pituitary peptide galanin (both at 200 nM) also increased the binding of [125I]GnRHa (30 pM) to anterior pituitary membranes, but to approximately one third of the extent produced by NPY. Salmon calcitonin, which, similar to NPY, is a strongly hydrophobic neuroendocrine peptide, did not alter GnRHa binding. Mg++ ion dose-dependently reduced the affinity of GnRHa binding, without changing the maximum binding capacity, and also attenuated the increase in GnRHa binding produced by NPY. To further characterize the nature of NPY interaction with anterior pituitary membranes, [125I] peptide YY was used to label NPY binding sites in anterior pituitary and hypothalamic membranes from ovariectomized rats. Specific, and predominantly high affinity, NPY binding sites were demonstrated in hypothalamic membranes, whereas NPY binding to anterior pituitary membranes could be resolved into high and low affinity components. These results show that at low nanomolar concentrations, NPY can enhance the association of GnRHa to receptors in anterior pituitary membranes. This demonstration of increased GnRHa binding in the presence of NPY may explain in part the action of this neuropeptide to potentiate GnRH-induced LH release from anterior pituitary cells.