Nitric oxide (NO), which was firstly identified as an endothelium-derived relaxing factor, has recently been demonstrated to be a neurotransmitter in the central and peripheral nervous systems. In the hypothalammus, abundant nitric oxide synthase (NOS) immunoreactivity and its histochemical marker, NADPH-diaphorase activity, have been demonstrated in the hypothalamo-neurohypophyseal system. In the present study, we examined whether NOS is coexpressed with posterior pituitary hormones in the rat hypothalamus by combination of oxytocin and vasopressin immunofluorescence and NADPH-diaphorase histochemistry. Most oxytocin-immunoreactive neurons in the paraventricular and supraoptic nuclei expressed NADPH-diaphorase activity, but virtually no vasopressin-immunoreactive neurons contained NADPH-diaphorase activity. This suggests that oxytocin neurons are the main source of NO production in the hypothalamic-pituitary system.