Nitric oxide is a free radical which is produced in the brain and is thought to be the first of a new class of neural messenger molecules. It is postulated to act by inducing an increase in cyclic guanosine monophosphate levels in target cells. The neuronal isoform of nitric oxide synthase, the enzyme responsible for the calcium-dependent synthesis of nitric oxide from L-arginine, has been purified from brain homogenate. Using a specific polyclonal antibody, we have localized brain nitric oxide synthase to the cytosol of discrete neuronal subpopulations and glial elements. These include non-pyramidal cells in the cerebral cortex, pyramidal and non-pyramidal cells of the hippocampus, aspiny neurons of the corpus striatum, basket, Purkinje and granule cells in the cerebellum and neurons of various brain stem nuclei. The localization of nitric oxide-producing neurons in morphologically different and neurochemically diverse cell types suggests a widespread neuromodulatory role for nitric oxide in the central nervous system of man.