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In the present study, we assessed the influence of monochloramine (NH2Cl) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in rat liver in vitro. When incubated with the partially purified cytosolic fraction from rat liver, NH2Cl (2.5–20 μM) dose-dependently enhanced XO activity concomitant with a decrease in XD activity, implying that NH2Cl can convert XD into the reactive oxygen species (ROS) producing form XO. The NH2Cl (5 μM)-induced XD/XO interconversion in the rat liver cytosol was completely inhibited when added in combination with an inhibitor of NH2Cl methionine (25 μM). A sulfhydryl reducing agent, dithiothreitol at concentrations of 0.1, 1 and 5 mM also dose-dependently reversed the NH2Cl (5 μM)-induced XD/XO interconversion. These imply that NH2Cl itself acts on the XD/XO interconversion, and that this conversion occurs at the cysteine residues in XD. Furthermore, using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate, it was found that NH2Cl could increase ROS generation in the cytoplasm of rat primary hepatocyte cultures, and that this increase might be reversed by an XO inhibitor, allopurinol. These results suggest that NH2Cl has the potential to convert XD into XO in the liver, which in turn may induce the ROS generation in this region.