Neuronal nitric oxide synthase (nNOS) is constitutively expressed by subpopulations of neurons in the CNS and is involved in neurotransmission, learning and memory, and neuronal injury. While the distribution of nNOS neurons has been characterized in the rodent CNS, the expression in human brain has not been well documented. We determined the expression of nNOS in second trimester human fetal and adult brain. In second trimester fetal brain, the nNOS neurons are concentrated in the developing cerebral cortex at the subplate zone and in layer VI, the striatum, and in certain brainstem nuclei. The nNOS neurons are sparsely distributed in the hippocampus, and virtually absent in the cerebellar cortex. The nNOS neurons in the subplate zone extend their processes radially, suggesting a developmental role, perhaps in guidance. The number and distribution of NADPH diaphorase-positive neurons corresponds to that of the nNOS neurons. While the distribution of nNOS neurons in the adult brain is similar to that found in fetal brain, the overall density is lower in the adult. The highest density of nNOS neurons is found in the striatum followed by the neocortex. A region-specific role for nNOS neurons in human brain and a potential developmental role for nNOS in the cerebral cortex are suggested by these data.