Nitric oxide (NO) has been implicated in the pathogenesis of brain injury from hypoxia-ischaemia. In the brain, the enzyme responsible for NO synthesis is neuronal nitric oxide synthase (nNOS). Using in situ hybridization, immunohistochemistry and NADPH diaphorase histochemistry, we examined the spatial and temporal expression of nNOS during development of the rat brain to determine whether the expression of nNOS delineates the areas of the brain that are selectively vulnerable to hypoxic-ischaemia injury. The expression of nNOS was localized to discrete areas of the brain. nNOS could be detected in the developing forebrain in the 10-day-old embryo (E10). From E14 to E18, the highest level of expression was in the cortical plate, where the majority of neurons were positive. However, this expression diminished with time; in the adult there were only a few nNOS-positive neurones in the deep layers of the cortex. Expression of nNOS was not detected prenatally in the basal ganglia. There was transient high-level expression during the first postnatal week. Thereafter, the basal ganglia exhibited the adult pattern of expression. Expression of nNOS could be detected in the hippocampus at E16. This expression remained constant with regional localization in layers CA1 and CA3 in the adult. Similarly, nNOS expression in the developing cerebellum was observed only after birth. From the first day after birth (P1) to P6, expression was limited to the molecular cell layer. As the cerebellum matured, nNOS expression could be detected in the inner granular layer. By P21, the adult distribution of nNOS expression was observed. All regions expressing nNOS mRNA also demonstrated nNOS protein expression and NADPH diaphorase catalytic activity. Our results demonstrate that nNOS expression in the developing brain correlates with regions of selective vulnerability to hypoxic-ischaemic injury, and, therefore, supports a role for NO in hypoxic-ischaemic injury in the developing brain.