The NADPH-diaphorase histochemical technique provides a simple and robust method to stain select populations of neurons throughout the brain. We have recently identified the enzyme responsible for this histochemical reaction to be nitric oxide synthase. This enzyme is responsible for the calcium-dependent synthesis of nitric oxide from arginine. Nitric oxide acts as a novel neural messenger by stimulating soluble guanylyl cyclase thereby increasing the levels of cyclic guanosine 3',5'-monophosphate in target cells. Thus the NADPH-diaphorase histochemical method allows the direct visualization of the neurons which use this novel signal transduction pathway. We now describe the detailed distribution of this enzyme in the rat brain. Our results suggest a widespread role for the nitric oxide-cyclic guanosine monophosphate system in the nervous system.