Expression and estrogen regulation of the genes for nitric-oxide (NO)-synthesizing enzymes (NO synthase, NOS) were investigated by in situ hybridization. This study focused on regions of the hypothalamus that contain estrogen receptors and regulate specific neuroendocrine functions related to female sexual behavior and food intake, among others. Ovariectomized (OVX) rats were treated with vehicle or 3 micrograms/100 g estradiol benzoate (EB) for 7 days. Brains were sectioned and hybridized with antisense riboprobes for neuronal NOS, macrophage NOS and endothelial NOS. In the hypothalamus, mRNA was clearly detectable only for the neuronal NOS with the probes used. A strong hybridization signal was observed in the supraoptic paraventricular and ventromedial nuclei (SON, PVN and VMN, respectively). Quantitative analysis showed an increase in neuronal NOS mRNA in the VMN of the OVX rats treated with EB. The increase was mainly in the ventrolateral aspect of the VMN. No significant changes were observed in the hypothalamic SON and PVN. The data suggest that the expression of neuronal NOS mRNA in VMN can be regulated by estrogen.