Ozone-induced lung injury in rats is focal, with the primary target sites being the distal trachea and the central acinus. In both area, ozone causes cellular injury and necrosis after short-term exposures, but the areas become tolerant to further injury after long-term exposure. To investigate the role of antioxidant enzymes in the resistance of the lung to injury from long-term ozone exposure, we measured activities of three antioxidant enzymes in airway samples microdissected from specific sites within the lung: distal trachea, lobar bronchi, major daughter axial bronchi, minor daughter bronchi, distal bronchiole, and parenchyma. Fischer 344 rats were exposed to 0, 0.5, and 1 ppm ozone 6 hr/day, 5 days/week for 20 months, or to 0, 0.12, and 1 ppm for 90 days. Glutathione transferase, glutathione peroxidase, and superoxide dismutase activities were measured at the end of the exposure periods. Data were normalized for DNA content (Units/mg DNA). For both the 90-day and 20-month exposures, the activities of all three enzymes were significantly elevated in a concentration-dependent fashion in the distal bronchioles. Compared to controls, animals exposed to 1.0 ppm ozone had superoxide dismutase activities 1.6x (90 days) and 2x (20 months) greater; glutathione peroxidase had activities 1.4x (90 days) and 1.6x (20 months) greater; and glutathione S-transferase had activities 1.5x (90 days and 20 months) greater. In animals exposed for 90 days, superoxide dismutase activity was lower in major daughter bronchi and greater in minor daughter bronchi and glutathione peroxidase activity was lower in major daughter bronchi. After 20 months of exposure, superoxide dismutase activity was significantly elevated in a dose-dependent fashion in the distal trachea; glutathione peroxidase activity decreased in the major daughter bronchi and increased in the minor daughter bronchi; and glutathione S-transferase activity decreased in the major daughter bronchi. There were no changes in antioxidant enzyme levels in other subcompartments. Superoxide dismutase activity increased in a concentration-dependent fashion in the whole lung homogenate of animals exposed for 90 days, but no differences were detected in whole lung homogenates of any other exposure groups. We conclude that (1) antioxidant enzyme activities are altered on a site-specific basis in response to long-term exposure to ozone; (2) the antioxidant enzymes respond differently in different lung subcompartments; (3) activities determined for the whole lung do not reflect changes in subcompartments with variable susceptibility to injury; and (4) changes in antioxidant enzyme activities are concentration-dependent and altered by length of exposure.