Peroxidation products formed from polyunsaturated lipids have DNA damaging potential. 4-oxo-2-hexenal(4-OHE), generated by the oxidation of ω-3 fatty acids, has been demonstrated to be mutagenic in vitro asassessed in the Ames test. To examine the carcinogenic risk of 4-OHE in vivo, initiation activity was investigatedin a five-week liver assay, established to be effective for screening of carcinogenic potential of mutagens. Sevenweek-old male F344 rats underwent two-thirds partial hepatectomy (PH) and were administered 4-OHEintragastrically at doses of 128, 80, 64, 40, 32, 20, or 0 mg/kg body weight (b.w.) at 18 hours thereafter, thenbeing fed on diet containing 0.015% 2-acetylaminofluorene from weeks 2 to 4. All rats were given with 0.8 ml/kgb.w. CCl4 at week 3. At week 5, all survivors were sacrificed and initiation activity was assessed with referenceto induction of glutathione S-transferase placental form (GST-P) positive foci in the liver. Mortality wassignificantly increased to 72.7% in the 128 mg/kg b.w. dose group compared with 0.9% in the control group.However, the average number of GST-P positive foci in the “128” dose group was 3.26±1.66 foci/cm2, notsignificantly different from the control value (2.78±1.33). Areas of GST-P positive foci were also similar (1.11±0.5and 1.53±1.33 mm2/cm2 in “128” and the control groups, respectively). These results showed 4-OHE to have nosignificant initiation activity in vivo.