Increases in acyl coenzyme A (CoA) oxidase activity due to peroxisome proliferation are postulated to cause oxidative stress via elevated production of H 2O 2, leading to DNA damage. These changes are suspected to be responsible for tumor formation caused by non-genotoxic carcinogens which do not bind to DNA but cause proliferation of peroxisomes. However, the activity of the peroxisomal enzyme acyl CoA oxidase assayed in vitro in the presence of excess fatty acyl CoA substrate may not reflect rates of H 2O 2 generation in intact liver where fatty acid supply is carefully controlled in part by delivery of substrate. The purpose of this work was to determine if rates of hepatic H 2O 2 generation were altered in perfused liver and in vivo following induction of H 2O 2-generating acyl CoA oxidase activity. Injection of the potent peroxisome proliferating agent perfluorooctanoate into rats 5 days prior to sacrifice caused an expected 4-fold increase of H 2O 2-generating acyl CoA oxidase activity measured in hepatic homogenates. In contrast, rates of H 2O 2 generation in perfused liver measured spectrophotometrically (660−640 nm) through a lobe of the liver were not altered by perfluorooctanoate treatment (7.3 ± 1.5 vs. 7.8 ± 0.5 μmol gh in livers from untreated control rats). Similar treatment with perfluorooctanoate also increased in vitro acyl CoA oxidase activity 9-fold in livers from deermice; however, rates of elimination of methanoi, a selective substrate for catalase in rodents whose oxidation is limited by the supply of H 2O 2, were not altered significantly in vivo (control, 110 ± 11 μmol gh vs. perfluorooctanoate, 112 ± 32 μmol gh ). Taken together, these data demonstrate that elevation of H 2O 2 formation by acyl CoA oxidase activity measured in vitro is not necessarily associated with increases in rates of H 2O 2 generation in intact perfused liver or in vivo, most likely due to rate-limitation in intact cells by fatty acid supply. These data do not support the hypothesis that the induction of peroxisomes leads to excessive H 2O 2 production and oxidative stress. It follows that alternative hypotheses to explain carcinogenesis caused by peroxisome-proliferating agents need to be considered.