The activity of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), an enzyme related to the synthesis of nitric oxide (NO), was studied histochemically in rat neocortex from the day of birth (P0) to young adulthood. At birth, NADPH-d containing neurons were already identifiable, sparsely distributed in the deep half of the pallium of the cerebrum. In addition, weakly stained bands of NADPH-d positive neuropil were detectable in layers VI and deep V and the cortical plate (CP). During the first postnatal week, NADPH-d positive neurons increased markedly, especially in CP and the superficial layers. By P7 the cells were mainly in layers VIb and II/III. Differential NADPH-d activity in the neuropil during this period appeared as higher activity gradually moving upwards until the highest intensity localised in layers II and upper III. In the caudal part of the cortex, the higher activity covered the whole of the supragranular layers. By the end of the second postnatal week, both the number and laminar distribution of NADPH-d neurons were adult-like, still mainly in layers VIb and II/III. The staining intensity in the neuropil was generally reduced but the banding pattern seen at P7 was still present. No detectable changes in the patterns of NADPH-d positive cells and neuropil in the neocortex occurred after the second postnatal week. The rostral part of the cortex matured slightly earlier than the caudal part. These results show that the development of NADPH-d activity correlates with the laminar differentiation and suggest that NADPH-d or NO may play an important role in the maturation of cortical neurons including the establishment of functional connections.