Pretreatment of rats with large doses of vitamin A (VA) potentiates the hepatotoxicity of CCl4. Because our previous studies indicate that VA treatment does not enhance CCl4 metabolism but does enhance CCl4-induced lipid peroxidation and activates liver Kupffer cells to release increased amounts of oxygen-centered free radicals, the current studies were designed to determine if VA treatment potentiates CCl4-induced liver injury through increased release of reactive oxygen species. Plasma clearance of colloidal carbon, an index of Kupffer cell phagocytic activity, was enhanced two- to threefold in rats treated for 7 days with VA (retinol, 250,000 IU/kg per day). Accordingly, VA treatment alone caused Kupffer cell activation. To determine if these activated Kupffer cells could potentiate hepatic injury through release of reactive oxygen species upon CCl4 challenge, polyethylene glycol coupled-superoxide dismutase (PEG-SOD, 10,000 IU/kg) or -catalase (PEG-CAT, 40,000 IU/kg) was given iv 2 hr after CCl4 (0.15 ml/kg, ip) to control or VA-pretreated rats to quench any released reactive oxygen. PEG-SOD and PEG-CAT effectively blocked VA potentiation of CCl4 liver injury as assessed at 24 hr by change in plasma ALT. Methylpalmitate (MP, 2 g/kg), an inhibitor of Kupffer cell phagocytosis and related oxygen burst, also blocked the potentiation of liver injury when given iv 24 hr before CCl4 to VA-pretreated rats. At the doses used, PEG-SOD or PEG-CAT did not influence CCl4 toxicity in control rats (at 0.15 or 2 ml CCl4/kg). Importantly, SOD, CAT, and MP blocked the enhanced lipid peroxidation induced by CCl4 in VA-pretreated rats. From these findings we conclude that the potentiation of CCl4 liver injury by VA pretreatment is mediated, at least in part, by active oxygen species released from Kupffer cells and possibly other macrophages that are activated by VA. Supporting this conclusion is the failure of VA pretreatment to increase the release of LDH from suspension of hepatocytes incubated with CCl4.