Antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferases are thought to be the primary cellular defense against reactive oxygen species. Since pulmonary injury produced by oxidant air pollutants like ozone is highly focal, involving primarily the trachea and centriacinar areas of the lung, measurements of alterations in antioxidant enzyme activities in whole lung may substantially underestimate changes occurring in target areas of the respiratory tract. We have applied a technique for preparation of lung specimens from well-defined anatomic locations to determine whether the focal injury associated with ozone exposure is related to an uneven distribution of antioxidant enzyme activity in the respiratory tract. Our study compared enzyme activities in rat and monkey, species which differ considerably in sensitivity to ozone-induced injury (monkey > rat). The activities of glutathione S-transferase varied less than twofold between different airway subcompartments for both the rat and monkey. Pulmonary veins had approximately 50% of the activity of airways in both species. Glutathione peroxidase activity was slightly higher in proximal compared to distal airways of the rat but was evenly distributed at all airway levels in the monkey. In both species, activity in pulmonary veins was lower than that in airways. The activity of superoxide dismutase was similar in rat and monkey and marked differences were not observed in the various subcompartments studied. Similarly, catalase activity was relatively evenly distributed in rat airways but, in the monkey, the distal bronchiole and lobar bronchus had marginally higher activity than the trachea. We conclude that: (1) measurement of antioxidant enzyme activities in anatomic subcompartments within the lung is feasible using microdissected specimens, (2) antioxidant enzyme activity can vary in different subcompartments of the lung of the same species, (3) the pattern of variation in enzyme activity differs by the enzyme and by species, and (4) species and subcompartment differences in ozone injury are not due primarily to differences in the distribution of antioxidant enzyme activity.