To understand the mechanism of peroxisome proliferator-induced oxidative stress in non-mutagenic carcinogenesis, the effect of ciprofibrate, a peroxisome proliferator, on the activities and protein amounts of various antioxidant enzymes in different subcellular compartments was examined. Ciprofibrate treatment for short-term (3 weeks) as well as long-term (12 weeks) duration increased the total cellular catalase activity, whereas superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities were decreased significantly. Withdrawal of ciprofibrate from the diet did not normalize these activities. The observed decreases in total cellular SOD and GPX activities following ciprofibrate treatment were due to significant decreases in cytosolic CuZn SOD and GPX, whereas mitochondrial levels of Mn SOD and GPX were relatively unchanged. The peroxisomal CuZn SOD and GPX activities were increased significantly after both short- and long-term treatment, whereas catalase activity was reduced. Western blot analysis of cytoplasm for GPX and CuZn SOD showed a significant decrease in GPX and CuZn SOD proteins. Mitochondrial GPX protein was found to be slightly decreased, whereas Mn SOD protein levels did not show any significant change. The excessive production of H2O2 by oxidases and O2- by the cytochrome P450 enzyme system, along with the observed loss of antioxidant protection by loss of activities of catalase in peroxisomes and GPX and CuZn SOD in cytoplasm, may be the critical factors in peroxisomal proliferator-induced oxidative stress and initiation and promotion of carcinogenesis by this class of non-mutagenic agents. Both enzyme activities, as well as protein amounts of GPX and CuZn SOD, were higher in peroxisomes but lower in cytoplasm in ciprofibrate-treated liver as compared to control liver. The Mn SOD protein was decreased in peroxisomes, whereas mitochondrial Mn SOD was relatively unaffected in ciprofibrate-treated liver as compared to control. These observations suggest that the regulation of expression of peroxisomal CuZn SOD and Mn SOD is different from their counterparts in other cellular compartments.