Nitric oxide (NO), a free radical gas that acts as a neural messenger, is produced from L-arginine by the enzyme nitric oxide synthase (NOS). The present study describes the autoradiographic localization throughout rat brain of the binding of [3H]L-NG-nitro-arginine ([3H]L-NOARG), a reversible NOS inhibitor, and confirms the heterogeneous distribution of the NOS enzyme. [3H]L-NOARG binding was enriched in discrete neuronal populations and the distribution observed was identical to that previously described for NOS using immunohistochemistry, in situ hybridization and NADPH-diaphorase staining. [3H]L-NOARG autoradiography should be a useful method for the further characterization and quantification of NOS in brain and other tissues.