Considerable evidence shows that gonadal steroids exert a facilitatory influence on levels and release of neuropeptide-Y (NPY) from the hypothalamus. However, it is not known whether gonadal steroids act directly on NPY-producing cells in the arcuate nucleus (ARC) of the hypothalamus to produce these facilitatory effects on NPY or whether they act on other cells that have a modulatory influence via synapses on ARC NPY cells. We applied the combined method of steroid autoradiography and immunocytochemistry to assess the localization of [³H]estradiol in relation to NPY-producing cells in the hypothalamus. Rats (n = 6) were bilaterally ovariectomized and injected intracerebroventricularly with colchicine. Twentyfour hours later each rat received an iv injection of 17β- [2,4,6,7)16,17-³H]estradiol (SA, 166 Ci/mmol) at a dose of 5.0 μg/kg BW. One hour after the injection of [³H]estradiol, the rats were perfused with 4% paraformaldehyde; brains were removed, frozen in isopentane precooled in liquid nitrogen (–190 C), sectioned, and processed for autoradiography. The autoradiograms were then incubated with specific antibodies for NPY immunostaining by the avidin-biotin-peroxidase method. The results revealed NPY-immunopositive cells in the ARC, striatum, hippocampus, amygdala, and cerebral cortex and a few cells in the median eminence. NPY-immunoreactive fibers were also detected in the internal layer of the median eminence. The largest number of neurons showing NPY immunoreactivity in the cytoplasm was detected in the ARC, and only in this nucleus did we observe colocalization of [³H]estradiol and NPY immunoreactivity in neurons. A population of NPY-immunopositive cells in the ARC (10-20%) exhibited nuclear [³H]estradiol; the majority of these cells were located in the lateral and ventral portions of the ARC. Since gonadal steroids stimulate hypothalamic NPY levels and release, this anatomical evidence of colocalization is suggestive of a direct genomic modulation of NPY neurosecretion by steroids in a subpopulation of hypothalamic NPY-immunopositive neurons. (Endocrinology127: 2752–2756, 1990)