This commentary focuses on the roles of CYP3A and CYP2E in alcohol-mediated increases in acetaminophen hepatotoxicity. CYP2E has been considered to be the main form of P450 responsible for such toxicity in animals and humans. However, CYP3A, which is also induced by alcohol, has been shown to have a greater affinity for acetaminophen than CYP2E. Previous experiments implicating CYP2E in alcohol-mediated increases in acetaminophen hepatotoxicity have used inhibitors of this form of P450 that are now proving to be non-specific. Triacetyloleandomycin (TAO) is a potent inhibitor of CYP3A that maintains specificity in vitro over a large concentration range. In rats treated with ethanol or the combination of ethanol and isopentanol, the major higher chain alcohol in alcoholic beverages, TAO protects animals from increases in acetaminophen hepatotoxicity, suggesting a major role of CYP3A. CYP2E may not have a major role due to the rapid loss of induced levels in the absence of continued exposure to ethanol. Knockout mice, which are being used to define the role of particular proteins in biological responses, have been developed for CYP2E1 and CYP1A2 but not CYP3A. Cyp2e1(-/-) and Cyp1a2(-/-) mice are more resistant to acetaminophen hepatotoxicity than wild-type strains, even though the amounts of the other forms of P450s are unaltered in the liver. These findings suggest that the relative amounts of P450s and not just kinetic characteristics determine their role in acetaminophen hepatotoxicity. The clinical implications of the findings that CYP3A can have a major role in acetaminophen-mediated hepatotoxicity are discussed.