In the present study, we investigated the mechanism of nitric oxide (NO) inactivation by endothelial cells. All experiments were performed in the presence of superoxide dismutase to minimize the peroxynitrite reaction. Incubation of the NO donor diethylamine/NO adduct with increasing amounts of intact cells led to a progressive decrease of the NO concentration, demonstrating a cell-dependent consumption of NO. In cell homogenates, consumption of NO critically depended on the presence of NADPH or NADH and resulted in the formation of nitrate. Both NO consumption and nitrate formation were largely inhibited by the heme poisons NaCN and phenylhydrazine as well as the flavoenzyme inhibitor diphenylene iodonium. Further characterization of this NO consumption pathway suggests that endothelial cells express a unique membrane-associated enzyme or enzyme system analogous to the bacterial NO dioxygenase that converts NO to nitrate in a NAD(P)H-, flavin- and heme-dependent manner.