NADPH diaphorase histochemistry selectively labels neurons in the nervous system. This technique has been used in many studies, but the function of the enzyme remains unclear. The authors characterized the enzyme biochemically and found that it is a nitric oxide synthase (NOS). NOS is a 150-kDa enzyme that converts L-arginine to nitric oxide (NO) with NADPH as a cofactor. NADPH diaphorase and NOS activities were found to copurify and both could be immunoprecipitated with an antibody specific for NADPH diaphorase. Additionally, NBT, a substrate for NADPH diaphorase, competitively inhibited NOS activity. These findings suggest that neuronal NADPH diaphorase is a nitric oxide synthase. NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide. The technique is based on the presence of an enzyme in certain neurons that catalyzes the NADPH-dependent conversion of a soluble tetrazolium salt to an insoluble, visible formazan. This method has been used to examine various populations of neurons in experimental and neuropathological studies. NADPH diaphorase-containing neurons appear relatively resistant to anoxia and excitotoxic damage. The enzyme has been well defined histochemically, but its function has remained a mystery. Previous attempts to characterize the enzyme biochemically have been hampered by the lack of a specific assay. The authors used a biochemical assay and an antibody specific for neuronal NADPH diaphorase to monitor the purification of the enzyme. NADPH diaphorase is an NADPH-dependent enzyme, so a purification protocol similar to that used for NOS was attempted. The results showed that NADPH diaphorase and NOS activities copurify and both could be immunoprecipitated. The purified protein was specifically detected in immunoblotting experiments. Both NOS and NADPH diaphorase activities could be immunoprecipitated with the NADPH diaphorase-specific antiserum but not with nonimmune rabbit serum. The results indicate that NADPH diaphorase is a neuronal nitric oxide synthase. Thus, the extensive literature on the histochemistry of NADPH diaphorase should be reexamined in light of this finding.NADPH diaphorase histochemistry selectively labels neurons in the nervous system. This technique has been used in many studies, but the function of the enzyme remains unclear. The authors characterized the enzyme biochemically and found that it is a nitric oxide synthase (NOS). NOS is a 150-kDa enzyme that converts L-arginine to nitric oxide (NO) with NADPH as a cofactor. NADPH diaphorase and NOS activities were found to copurify and both could be immunoprecipitated with an antibody specific for NADPH diaphorase. Additionally, NBT, a substrate for NADPH diaphorase, competitively inhibited NOS activity. These findings suggest that neuronal NADPH diaphorase is a nitric oxide synthase. NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide. The technique is based on the presence of an enzyme in certain neurons that catalyzes the NADPH-dependent conversion of a soluble tetrazolium salt to an insoluble, visible formazan. This method has been used to examine various populations of neurons in experimental and neuropathological studies. NADPH diaphorase-containing neurons appear relatively resistant to anoxia and excitotoxic damage. The enzyme has been well defined histochemically, but its function has remained a mystery. Previous attempts to characterize the enzyme biochemically have been hampered by the lack of a specific assay. The authors used a biochemical assay and an antibody specific for neuronal NADPH diaphorase to monitor the purification of the enzyme. NADPH diaphorase is an NADPH-dependent enzyme, so a purification protocol similar to that used for NOS was attempted. The results showed that NADPH diaphorase and NOS activities copurify and both could be immunoprecipitated. The purified protein was specifically detected in immunoblotting experiments. Both NOS and NADPH diaphorase activities could be immunoprecipitated with the NADPH diaphorase-specific antiserum but not with nonimmune rabbit serum. The results indicate that NADPH diaphorase is a neuronal nitric oxide synthase. Thus, the extensive literature on the histochemistry of NADPH diaphorase should be reexamined in light of this finding.