Based on previous studies demonstrating that reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (ND) activity is modulated by estradiol and the discovery of a new subtype of estrogen receptor highly expressed in the paraventricular nucleus of the hypothalamus (PVN), a possible estrogen influence on this activity was investigated in the neuronal populations (magno- and parvicellular) of this nucleus. Cryostat sections were cut and processed for the histochemical detection of the ND activity. Following ovariectomy (14 days), numerical data displayed a slight decrease in the number of ND-neurons, especially in the posterior magnocellular and the medial parvicellular subdivisions, which was reversed after daily treatment with estradiol benzoate. Administration of estradiol benzoate to male rats (14 days) induced a significant increase (P < 0.05) in the number of ND-neurons, mainly at the level of the posterior magnocellular subdivision. These data indicate that paraventricular ND-neurons are influenced by estradiol.