To determine if nitrogen dioxide (NO2), a gaseous free radical, modifies the protective antioxidant pool present in respiratory tract lining fluids, a random, double-blind study utilizing flexible fiberoptic bronchoscopy with bronchial and bronchoalveolar lavage was performed. Healthy, nonsmoking, asymptomatic subjects were exposed to filtered air and 2 ppm NO2 for 4 h on separate occasions. To examine the kinetics of the NO2-induced antioxidant reactions, 44 subjects were randomly assigned to one of three groups. Bronchoscopy was performed 1.5 h (group 1), 6 h (group 2) or 24 h (group 3) after each exposure. Reduced glutathione (GSH), uric acid, and ascorbic acid concentrations were determined in both bronchial and bronchoalveolar lavage fluid fractions. In addition, bronchoalveolar lavage fluid was screened for malondialdehyde as a marker of lipid peroxidation. Exposure to NO2 resulted in a rapid (1.5 h) loss of uric acid from the bronchial region, however by 6 h after exposure it had increased significantly above control uric acid concentration in this region. At 24 h after exposure, uric acid concentration had returned to the control level. A similar response of uric acid to NO2 was seen in the bronchoalveolar region. Ascorbic acid was also decreased in bronchial and bronchoalveolar lavage fluids 1.5 h after exposure to NO2, but returned to control values by 6 h. In marked contrast, significant increases in GSH concentration were seen at 1.5 and 6 h in bronchial lavage fluid after exposure to NO2, which subsequently returned to control levels by 24 h. No change in bronchoalveolar lavage fluid GSH concentration or malondialdehyde content was seen after NO2 exposure. These data support the view that antioxidants present in lung fluids react with, and hence modulate the impact of, NO2 on the lung.