The regional distribution of nitric oxide synthase in the central nervous system was assessed by quantitative autoradiography using [3H]L-NG-nitro-arginine binding in wild-type mice (SV-129 and C57black/6) and in mice lacking expression of the neuronal (type 1) and endothelial (type 3) nitric oxide synthase gene. The distribution of nitric oxide synthase binding sites in wild-type mice was similar to that described for rat brain by nicotinamide adenine dinucleotide phosphate-diaphorase staining and immunohistochemistry, and as determined by quantitative autoradiography. In the wild-type mice, the densest labelling was observed in the granular layer of the olfactory bulb, tenia tecta, rhinal fissure, amygdaloid complex and molecular layer of cerebellum. The islands of Calleja, the hippocampal CA1 and CA3 subfields, dentate gyrus, cortical layers I-II, the superficial gray layer of superior colliculus and the granule layer of cerebellum displayed intermediate binding. Cortical layers III-VI, the striatum and the thalamus were only weakly labelled. Binding was saturable and of high affinity, and was displaced by 7-nitroindazole (100 microM), a potent and selective inhibitor of type 1 nitric oxide synthase, and by unlabelled L-NG-nitro-arginine (10 microM). The density of [3H]L-NG-nitro-arginine binding was dramatically reduced in all brain regions in type 1 mutant mice, whereas there were no detectable binding differences between wild-type and type 3 nitric oxide synthase mutant mice. Hence, type 1 nitric oxide synthase is the major source of [3H]L-NG-nitro-arginine binding in the mouse brain. [3H]L-NG-Nitro-arginine autoradiography may be a useful tool to quantify nitric oxide synthase in different brain areas after pharmacological or physiological manipulations.