Pretreatment of cultured rat hepatocytes with ethanol alone or in combination with isopentanol, the major higher chain alcohol in alcoholic beverages, significantly increased CYP3A and acetaminophen (APAP) bioactivation, with no increase in APAP toxicity. Caffeine has previously been shown to activate CYP3A activity in vitro and to increase APAP hepatotoxicity in rodents pretreated with prototypic inducers of CYP3A. Here we found that caffeine enhanced APAP toxicity in cultured rat hepatocytes pretreated with the alcohols. The caffeine-mediated increase in APAP toxicity was similar in cells treated with ethanol or isopentanol alone or in combination. These findings suggest that even small increases in CYP3A are sufficient to support caffeine-enhanced APAP toxicity. Triacetyloleandomycin inhibited CYP3A activity in intact hepatocytes and protected alcohol-pretreated cells from caffeine enhancement of APAP toxicity. This protection was associated with decreased formation of the toxic metabolite of APAP. The results indicate that CYP3A is responsible for the caffeine-mediated stimulation of APAP toxicity. Our results suggest that caffeine may be an additional risk factor for developing alcohol-mediated APAP hepatotoxicity.