Recent pharmacological studies have suggested a role for neuropeptide Y (NPY) in control of LH secretion. The present study examined the effects of estradiol benzoate (EB) given alone or in combination with progesterone (P), on the concentrations of immunoreactive NPY in microdissected nuclei of the rat brain, in association with changes in LHRH concentrations and LH release. Forty-eight hours after the administration of EB to ovariectomized rats, serum LH was significantly reduced. Concentrations of NPY, measured by a sensitive and specific RIA, were also reduced in the median eminence, arcuate nucleus, and interstitial nucleus of the stria terminalis. As expected, administration of P to these EB-primed rats induced a sequential rise and fall of LHRH in the median eminence, followed by a marked LH surge in the afternoon. Interestingly, NPY concentrations in the median eminence also increased and then decreased after P, with a time course similar to that shown by LHRH. In the arcuate nucleus and interstitial nucleus of the stria terminalis, P treatment did not affect NPY concentrations further. Sequential EB plus P treatment significantly reduced NPY levels in the medial preoptic nucleus compared to oil vehicle controls. Levels of immunoreactive NPY in the ventromedial nucleus and periventricular nucleus were largely unaffected by ovarian hormone treatments. These results indicate that ovarian steroids, which modulate LH secretion, affect NPY concentrations in those specific areas of the brain which are known to be innervated by the LHRH neurons. These observations support the hypothesis that NPY may participate in the neural regulation of LHRH and LH secretion.