4-(N,N-Diacetylamino)benzo[a]pyrene, a potent mutagen, is derived from a carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene. Metabolism of this compound by rat liver microsomes was studied. Metabolites were separated by reversed-phase high-performance liquid chromatography and were identified by analysis of their UV-vis absorption, mass, and proton nuclear magnetic resonance spectral data. Seven metabolites were identified, namely, the 9-phenol, 1,9-hydroquinone, and trans-9,10-dihydrodiol of 4-(N,N-diacetylamino)benzo[a]pyrene, 4-(N-acetylamino)benzo[a]pyrene, and the 5-phenol, 5,9-hydroquinone, and trans-9,10-dihydrodiol of 4-(N-acetylamino)benzo[a]pyrene. Comparison of these results with those of metabolism of benzo[a]pyrene indicates that the N,N-diacetylamino substitutent at the 4-carbon of benzo[a]pyrene inhibits metabolism at the peri position (3-carbon) and positions (6-, 7-, and 8-carbons) remote from the substituent. The results also indicate that while 4-(N,N-diacetylamino)-benzo[a]pyrene is a substrate of the rat liver microsomal deacetylase, the formed 4-(N-acetylamino)benzo[a]pyrene apparently is not a substrate.