In vitro effects of acetone on cytochrome P450 (P450)-dependent benzo(a)pyrene (B(a)P) hydroxylation supported by cumene hydroperoxide (Cu00H) or NADPH/O₂ systems were studied using 3-methylcholanthrene-pretreated rat liver microsomes. The maximal rate of B(a)P hydroxylation at constant concentration (80 μM) of the substrate was observed in the presence of 30 μM Cu00H. However, at concentrations higher than 30 pM CuOOH the hydroxylation rates were rapidly decreased. In contrast to CuOOH, at a concentration of 200 p.M NADPH, B(a)P hydroxylation rate reached a plateau. At concentrations higher than 200 μM NADPH, the rates of substrate hydroxylation were maintained at the maximal rate with no inhibition. Acetone at 1 % (v/v) enhanced both CuOOH- and NADPH/O₂-supported B(a)P hydroxylation at the optimal concentrations of the cofactors. At concentrations higher than 1 % (v/v) acetone, substrate hydroxylation was sterero specific under the support of these two cofactors; it was strongly enhanced with 30 μM Cu00H, but rather inhibited in the 200 μM NADPH/O₂ system. The lipid peroxidation rate induced during CuOOH-supported P450-dependent B(a)P hydroxylation was increased as CuOOH concentrations were increased. Acetone in the concentration range of 2.5-7.5%(v/v) inhibited lipid peroxidation during CuOOH supported B(a)P hydroxylation. The finding that CuOOH-supported B(a)P hydroxylation is greatly enhanced by acetone suggests that acetone may contribute more to the activation of oxygen (for the insertion of oxygen into the substrate) in the presence of CuOOH than with NADPH/O₂. Acetone may also contribute to the partial inhibition of destruction of microsomal membranes by lipid peroxidation.