This study characterizes the developmental expression of NADPH-diaphorase from embryo to adulthood in the forebrain, midbrain and cerebellum of rat brain via histochemical staining. On embryonic day 12 no neurons stained. Labeling was observed in certain nuclei from E15 through the postnatal period to adulthood. Labeling in neurons increased or maintained a constant level with increased age. The embryo demonstrated substantial labeling in neurons of the caudate putamen, bed nucleus of the stria terminalis, preoptic area, lateral hypothalamic area, paraventricular thalamic nucleus, ventromedial hypothalamic nucleus, magnocellular nucleus posterior commissure, and periaqueductal central gray. Additional neuronal labeling was observed postnatally in the olfactory bulb, cerebral cortex, amygdala, various nuclei of the thalamus, interpeduncular nucleus, linear nucleus of the raphe, pretectal area and superior colliculus. In the cerebellum, labeling appeared only after P14 in cells of the molecular cell layer and granular cell layer. The sizes of labeled neurons developed significantly from P4 to P14 in several nuclei. The distinctive temporal and spatial expression pattern of NADPH-diaphorase implies that the NO/cGMP system may play an important role in physiological and developmental functions.