To clarify the role of nitric oxide (NO) in the ontogeny of arginine vasopressin (AVP) and oxytocin (OXT) neurons in the hypothalamo-neurohypophysial system (HNS), we observed the coexpression pattern of NADPH-diaphorase (NADPH-d) activity and AVP- or OXT-immunoreactivity (IR) in the rat hypothalamus and posterior pituitary during the postnatal period. The enzymatic activity of NADPH-d was observed in the supraoptic nucleus (SON), paraventricular nucleus (PVN), median eminence (ME) and posterior pituitary throughout the postnatal development. AVP-containing neurons were clearly observed from postnatal day 1 in both the SON and PVN, while OXT-containing neurons were recognized from postnatal day 14. The coexistence of NADPH-d and AVP or OXT was detected in the SON from postnatal day 14. At postnatal day 21, the coexpression pattern was approximately the same as that of the SON and PVN in adult rats. Our findings indicated that the expression of NADPH-d and OXT was observed from almost the same postnatal period in both the SON and PVN. In addition, the pattern of increased numbers of NADPH-d positive fibers was similar to that of OXT-immunoreactive fibers in both the inner layer of the ME and the posterior pituitary. A good correlation was thus obtained between OXT expression and NADPH-d activity in the HNS during postnatal development. The present study suggests that NO is more closely involved in the expression and regulation of secretion of OXT than AVP.