Vasoactive intestinal peptide (VIP) inhibits pulsatile luteinizing hormone (LH) secretion in the ovariectomized rat. Hypothalamic nuclei known to contain VIPergic neurons were destroyed electrolytically to determine whether either an increased response or a loss of response to exogenous VIP would result. Bilateral electrolytic lesions were made of either the suprachiasmatic (SCN) or paraventricular (PVN) nuclei in separate experiments; all animals received an intracerebroventricular cannula at the same time. Sham-lesioned animals were used as a control. One week later, a catheter was placed in the jugular vein of each rat and, after a recovery period of at least 2 h, blood samples were taken every 5 min for 3 h. After a control period of 1.5 h, either VIP or saline was infused into the third ventricle for an additional 1.5 h. Two doses of VIP were used: 3.5 nmol/h, previously shown to be inhibitory, and 0.4 nmol/h, which is ineffective in ovariectomized rats. LH was measured in the plasma by radioimmunoassay. The high dose of VIP lowered mean LH levels and pulse frequency but had no effect on pulse amplitude in both sham-lesioned and SCN-lesioned rats. The low dose of VIP did not affect pulsatile LH patterns in sham-lesioned rats, but did lower mean LH and pulse frequency in SCN-lesioned rats, indicating a denervation hypersensitivity subsequent to SCN lesions. Destruction of the PVN abolished the inhibitory effect of the high dose of VIP on pulsatile LH release. The low dose of VIP was ineffective in both PVN- and sham-lesioned animals. We interpret these results to mean that exogenous VIP inhibits pulsatile LH secretion in the ovariectomized rat through an effect on the terminal fields in the PVN of VIPergic neurons whose cell bodies are in the SCN, and that this effect is mediated by a non-VIPergic connection between the PVN and the gonadotropin-releasing hormone pulse generator.