The study by Hope et al. investigates the biochemical function of neuronal NADPH diaphorase, a widely used histochemical marker for neurons. The researchers purified NADPH diaphorase from rat brain using affinity chromatography and anion-exchange HPLC, and found that it copurified with nitric oxide synthase, a 150-kDa enzyme responsible for converting L-arginine to citrulline and nitric oxide. Both activities were immunoprecipitated with an antibody specific for neuronal NADPH diaphorase and competitively inhibited by the substrate nitro blue tetrazolium. This suggests that neuronal NADPH diaphorase is indeed a nitric oxide synthase, providing a specific histochemical marker for neurons producing nitric oxide. The findings highlight the importance of re-examining the literature on NADPH diaphorase histochemistry in light of its role in nitric oxide production in the nervous system.The study by Hope et al. investigates the biochemical function of neuronal NADPH diaphorase, a widely used histochemical marker for neurons. The researchers purified NADPH diaphorase from rat brain using affinity chromatography and anion-exchange HPLC, and found that it copurified with nitric oxide synthase, a 150-kDa enzyme responsible for converting L-arginine to citrulline and nitric oxide. Both activities were immunoprecipitated with an antibody specific for neuronal NADPH diaphorase and competitively inhibited by the substrate nitro blue tetrazolium. This suggests that neuronal NADPH diaphorase is indeed a nitric oxide synthase, providing a specific histochemical marker for neurons producing nitric oxide. The findings highlight the importance of re-examining the literature on NADPH diaphorase histochemistry in light of its role in nitric oxide production in the nervous system.