Xanthine oxidase (XO) was found to convert nitric oxide (NO.) released from spermine-NONOate to nitroxyl (HNO), the one-electron reduction product of NO., in the presence of its substrate hypoxanthine under anaerobic conditions. Under these conditions, XO lost its activity. Upon aerobic incubation of XO with its substrate, neither conversion of NO. to HNO nor inactivation of the enzyme was observed. Angeli's salt (an HNO generator) or synthetic peroxynitrite inactivated XO at low concentrations, whereas high concentrations of diethylamine-NONOate (an NO. donor) and SIN-1 (which generates peroxynitrite by releasing both NO. and superoxide) were required to inactivate XO. These results suggest that HNO generated by XO under anaerobic conditions inactivates XO. As both XO and NO. synthase are activated and/or induced in ischemia-reperfusion injury, HNO formed by XO may contribute to pathogenesis by exerting its potent oxidation activity against a variety of biological compounds. (C) 2004 Elsevier Inc. All rights reserved.