The distribution of nitric oxide synthase (NOS), the enzyme which produces nitric oxide, has previously been studied in the rat central nervous system (CNS) using the NADPH-diaphorase technique and anti-NOS antibodies. However, the former method may not always be selective for NOS while the latter is not quantitative. Therefore a selective, quantifiable method would be desirable. L-NG-Nitro-arginine, an inhibitor of NOS, is available in a tritiated form which we have shown to bind to NOS. We have now examined the regional distribution of [3H]L-NG-nitro-arginine binding in the rat CNS using autoradiography. [3H]L-NG-nitro-arginine specific binding was seen in a number of brain regions with the highest levels in the accessory olfactory bulb, the amygdaloid complex, the Islands of Calleja and the cerebellum. This regional distribution of [3H]L-NG-nitro-arginine binding sites in the rat CNS was, in general, similar to that seen with the NADPH-diaphorase method and anti-NOS antibodies, consistent with the view that all three methods identify NOS in the CNS. Thus, [3H]L-NG-nitro-arginine appears to be a useful radioligand for studying the distribution of NOS in the CNS as its binding is quantifiable and apparently selective for NOS.