This study was carried out to clarify the central effects of neuropeptide Y (NPY) on gonadotropin and prolactin (PRL) release in the male rat and to evaluate the hypothesis that it may play a role in negative feedback regulation of gonadotropin secretion in the male. NPY (4 µg in 2 µl 0.9% NaCl) or an equal volume of saline was microinjected into the third cerebral ventricle of conscious, unrestrained rats and its effects on plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and PRL cencentrations, and the gonadotropin response to LH-releasing hormone (LHRH) were measured by radioimmunoassay in noncastrated, castrated (3 days before the experiment) rats or animals treated with flutamide, an androgen receptor blocker (10 mg/kg b.w. per day subcutaneously for 3 days). Blood samples were drawn periodically from a Silastic catheter in the external jugular vein just before and for 120 min after intraventricular injections. At 90 min after injection 25 ng of LHRH were infused intravenously and 3 additional blood samples were withdrawn at 10-min intervals after the injection. Normal male rats responded to NPY with a decrease in plasma LH concentrations at 60 min. Plasma LH was significantly lower at 120 min than values in the saline-injected controls. At that time there was a significant 2-fold augmentation of their response to LHRH. In contrast, NPY elevated plasma FSH significantly versus values in controls at 15 and 30 min after injection, but the FSH release in response to LHRH was not altered. In castrated males NPY caused a rapid decline of their elevated plasma LH levels. At 60 min after NPY injection the mean plasma LH concentration was 22% of the initial level; meanwhile, plasma FSH levels were not significantly changed. The castrates displayed no change in LH responsiveness to LHRH as compared to the response of saline-injected control rats, but had a highly significant increase in FSH responsiveness. In flutamide-treated animals which had slightly elevated initial plasma LH values, NPY altered the plasma LH concentrations in a similar manner to those of normal rats; however, there was a decreased response to LHRH, and plasma FSH and responsiveness of FSH to LHRH were unaltered. At 30 min after NPY injection PRL levels decreased significantly in all experimental groups and remained low for the duration of the experiment. The mean area under the plasma PRL concentration curves for the 2-hour duration of the experiment relative to the control values was 44% in normal rats and 64% in the castrates. The results of this work demonstrate the suppressive effect of this dose of NPY injected into the third cerebral ventricle on LH and PRL release which was accompanied by a sensitization of the response of LH to LHRH in normal rats only and an increase in plasma FSH in normals and an increased response of FSH to LHRH in castrate animals only. Because NPY mimics the action of androgens on LHRH release, we suggest that it may be involved in negative feedback regulation of LHRH release in male rats.