An initiation-promotion protocol was used to test the hypothesis that unleaded gasoline (UG) vapor acts as a liver tumor promoter in female mice under exposure conditions in which UG was hepatocarcinogenic in a cancer bioassay. Twelve day old female B6C3F1 mice were injected with N-nitrosodiethylamine (DEN, 5 mg/kg, i.p.) or vehicle. Starting at 5–7 weeks of age, mice were exposed by inhalation 6 h/day, 5 days/week for 13 weeks to 0 or 2039 p.p.m. of PS-6 blend UG, the same gasoline blend used in the cancer bioassay. Putative preneoplastic lesions in liver, characterized mainly as basophilic foci in H&E-stained liver sections, were found exclusively in mice treated with DEN. While similar numbers of altered hepatic foci were found in DEN-initiated mice treated with 0 or 2039 p.p.m. UG, UG treatment significantly increased both the mean volume (3.2-fold) and the volume fraction (3.6-fold) of the foci. To determine if UG induced CYP2B, a subfamily of cytochrome P450 commonly induced by liver tumor promoters in rodents, pentoxyresorufin-O-dealkylase (PROD) activity was assayed in hepatic microsomes derived from the above livers. UG vapor increased hepatic PROD activity ∼8-fold, while increasing cytochrome P450 content only ∼30%. To ascertain if a more recent blend of UG, API 91-1, would have similar biological effects as PS-6, female B6C3F1 mice were gavaged for 3 days with corn oil or 1800 mg/kg/day PS-6 or API 91–1 blend UG. PS-6 and API 91-1 blend UG induced similar increases in relative liver weight (∼25%), PROD activity (∼9-fold) and hepatocyte labeling index (∼8-fold) relative to controls. These data demonstrate that PS-6 blend UG vapor promotes preneoplastic lesions and induces CYP2B in female mouse liver under exposure conditions in which it causes liver tumors, and suggest that a more recent blend of UG may have similar effects.