The existence of a relationship between clofibrate-induced peroxisome proliferation and oxidative stress mediated by activated oxygen species was studied in intact peroxisomes purified from Pisum sativum L. plants. Incubation of leaves with 1 mM clofibrate produced a remarkable increase in the peroxisomal activity of acyl-CoA oxidase and, to a lesser extent, of xanthine oxidase, whereas there was a nearly complete loss of catalase activity and a decrease in Mn-superoxide dismutase. Ultrastructural studies of intact leaves showed that clofibrate induced a five- and twofold proliferation of the peroxisomal and mitochondrial populations, respectively, in comparison with those in control leaves. Prolonged incubation with clofibrate produced considerable alterations in the ultrastructure of cells. In peroxisomal membranes, the NADH-induced generation of O2- radicals, as well as the lipid peroxidation of membranes, increased as a result of treatment of plants with clofibrate. In intact peroxisomes treated with this hypolipidemic drug, the H2O2 concentration was higher than in peroxisomes from control plants. These results demonstrate that clofibrate stimulates the production of activated oxygen species (O2- and H2O2) inside peroxisomes, as well as the lipid peroxidation of peroxisomal membranes. This effect is concomitant with a decrease of catalase and Mn-SOD activities, the main peroxisomal enzymatic defenses against H2O2 and O2-, and indicates that in the toxicity of clofibrate, at the level of peroxisomes, an oxidative stress mechanism mediated by activated oxygen species is involved.