The widely used NADPH-diaphorase reaction for demonstrating neuronal nitric oxide synthase is not as specific as previously thought, as it visualizes both a nitric oxide synthase-related activity and a nitric oxide synthase-unrelated diaphorase. In the present study, we used the rat olfactory bulb as a model to characterize the NADPH-diaphorase activity of neuronal nitric oxide synthase histochemically in comparison with neuronal nitric oxide-unrelated diaphorase activity. The NADPH-diaphorase activity of nitric oxide synthase peaked at pH 8 and at Triton X-100 concentrations of 1-2.5%. It was stable in an acidic environment but was reduced in the presence of Triton X-100 and was inactivated by the flavoprotein inhibitor, diphenyleneiodonium. It preferred beta-NADPH as the co-substrate to alpha-NADPH and alpha-NADH. In contrast, nitric oxide synthase-unrelated diaphorase peaked at pH 10, displayed a Triton X-100 optimum at a concentration of 1%, was unstable in an acidic environment and used beta-NADPH, alpha-NADPH and alpha-NADH to similar extents. Differences in the characteristics between neuronal nitric oxide synthase-related and nitric oxide synthase-unrelated NADPH-diaphorase can be used to increase the specificity of the histochemical nitric oxide synthase marker reaction.