Abstract: 2-hydroxy-1,4-naphthoquinone (HNQ; Lawsone, CAS 83-72-7) is the main dye ingredient found in the natural plant of Henna (Lawsonia inermis). The percentages of superoxide anion (O2.) and hydrogen peroxide (H2O2,) formation during the oxidation of 100 μM phenanthridine by guinea pig aldehyde oxidase have been measured and found to be 6-10% and 85-90%, respectively. The effect of HNQ on the initial rates of phenanthridine oxidation, hydrogen peroxide formation and superoxide anion with aldehyde oxidase was investigated and compared with those of xanthine oxidase. It was found that HNQ is a specific inhibitor of aldehyde oxidase in vitro. In this study, HNQ inhibits the production of superoxide anion and substrate oxidation more potently than hydrogen peroxide. Thus, the site of interaction is thought to be flavin semiquinone, FADH.. The IC50 value of HNQ with phenanthridine oxidation by aldehyde oxidase is 9.3±1.1 μM, which in excess of 15 fold of maximal plasma concentrations (Cmax) of HNQ, indicating a high degree of safety margin. The interaction of HNQ with aldehyde oxidase has been also investigated using different electron acceptors including, oxygen, potassium ferricyanide and cytochrome c and found to be equipotent.