Nitrogen dioxide (NO2) is an environmental oxidant that causes acute lung injury. Absorption of this aqueous insoluble gas into the epithelial lining fluid (ELF) that covers air space surfaces is, in part, governed by reactions with ELF constituents. Consequently, NO2 absorption is coupled to its chemical elimination and the formation of ELF-derived products. To investigate mechanisms of acute epithelial injury, we developed a model encompassing the spatial arrangements of the lung surface wherein oxidation of cell membranes immobilized below a chemically defined aqueous compartment was assessed after NO2 exposures. Because aqueous-phase unsaturated fatty acids displayed minimal NO2 absorptive activity, these studies focused on glutathione (GSH) and ascorbic acid (AH2) as the primary NO2 absorption substrates. Results demonstrated that membrane oxidation required both gasphase NO2 and aqueous-phase GSH and/or AH2. Membrane oxidation was antioxidant concentration and exposure duration dependent. Furthermore, studies indicated that GSH- and AH2-mediated NO2 absorption lead to the production of the reactive oxygen species (ROS) O-2. and H2O2 but not to .OH and that Fe-O2 complexes likely served as the initiating oxidant. Similar results were also observed in combined systems (GSH + AH2) and in isolated rat ELF. These results suggest that the exposure-induced prooxidant activities of ELF antioxidants generate extracellular ROS that likely contribute to NO2-induced cellular injury.