The distal trachea and centriacinus of the lung are primary sites of acute injury during short-term ozone exposure; long-term exposure yields cells in these areas that are resistant to high doses of oxidant gases. Epithelial cells located in primary sites for ozone injury are also targets for chemicals that undergo cytochrome P450 (CYP)-dependent activation. These studies were designed to compare the effects of ozone exposure on pulmonary CYP2E1 in susceptible and nonsusceptible sites within the airway tree of lung. CYP2E1 activity was measured in well-defined regions of airways using p-nitrophenol, a CYP2E1-selective substrate, with HPLC/ electrochemical detection of the p-nitrocatechol. Alterations in distribution of CYP2E1 were evaluated by immunohistochemistry. CYP2E1 activities were highest in the distal bronchioles and minor daughter airways but were much lower in the lobar bronchi/ major daughter airways and trachea. Immediately after short-term ozone exposures (8 h, 1 ppm), CYP2E1 activities were elevated only in the lobar bronchi/major daughter airways. These activities remained above the filtered air control at 1 day but returned to control levels by 2 days. Immunohistochemical assessment of CYP2E1 protein in ozone and filtered air-exposed animals was consistent with the activity measurements. After long-term ozone exposures (90 days, 1 ppm), CYP2E1 activities were decreased in the major and minor daughter airways. These studies indicate that CYP2E1 activities vary substantially by airway level. However, ozone exposure only results in minimal alterations in activity with varying concentration of ozone, length of exposure, and time after exposure in any of the lung subcompartments examined.