The hepatic tumor promoting activity of the planar 0–1 ortho (~9.7 /w) and the nonplanar 2–4 ortho (~90.3 /w) fraction of the commercial PCB mixture Aroclor 1260 was studied using a medium-term two-stage initiation/promotion bioassay in female Sprague–Dawley rats. Fractionation was carried out on an activated charcoal column. The composition of the effluent from the column was tested by GC–ECD. The absence of planar compounds in the 2–4 ortho fraction was confirmed by GC–MS analysis. The dioxin-like toxic potency of the fractions was determined with the DR-CALUX assay. The animal experiment was started with the initiation procedure (diethylnitrosamine injection, 30 mg/kg body wt ip, 24 h after 2/3 hepatectomy), followed 6 weeks later by the promotion treatment, which consisted of a weekly subcutaneous injection during 20 weeks. Exposure groups (n = 10) received the following treatments (dose/kg body wt/week): Aroclor 1260 (10 mg), 0–1 ortho fraction (0.97 mg), 2–4 ortho fraction (1, 3, or 9 mg), a reconstituted 0–4 ortho fraction (9.97 mg), 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153; 1 or 9 mg), 2,3,7,8-TCDD (1 g; positive control) or corn oil (1 ml; vehicle control). One group did not receive a promotion treatment. All exposure groups exhibited a significantly increased volume fraction of the liver occupied by hepatic foci positive for the placental form of glutathione-S-transferase-p compared to the corn oil control, except for the groups treated with 0–1 ortho fraction and 1 mg PCB 153/kg body wt/week. Approximately 80␘f the total tumor promoting capacity of the reconstituted 0–4 ortho fraction could be explained by the 2–4 ortho PCB fraction while the 0–1 ortho fraction had only a negligible contribution. These results suggest that the majority of the tumor promotion potential of PCB mixtures resides in the non-dioxin-like fraction, which is not taken into account in the toxic equivalency factor (TEF) approach for risk assessment of PCBs. This may result in an underestimation of the tumor promotion potential of environmental PCB mixtures.