In the developing cerebral cortex, neuronal nitric oxide synthase (nNOS) is expressed abundantly, but temporarily. During the early postnatal stage, cortical neurons located in the multi-layered structure of the cortical plate start forming well-organized cortical circuits, but little is known about the molecular machinery for layer-specific circuit formation. To address the involvement of nitric oxide (NO), we utilized a new NO indicator (DAR-4M) and developed a protocol for the real-time imaging of NO produced in fresh cortical slices upon N-methyl-D-aspartic acid stimulation. At postnatal day 0 (P0), NO production was restricted to the deep layers (layers V and VI) of the somatosensory cortex where transient synapses are formed. At P10, the production of NO was expanded to layer IV where large numbers of thalamocortical axons form synapses. The pattern of NO production could correspond to active sites for synaptic formation. This study is the first clear demonstration of NO production in the postnatal mouse neocortex. The findings presented may reflect a function of NO in relation to the layer-specific development of neural circuits in the neocortex.