Metabolism of 7-chlorobenz(a)anthracene (7-Cl-BA) by liver microsomes from untreated rats, and from rats pretreated with either 3-methylcholanthrene or phenobarbital was studied. The metabolites were isolated by HPLC and characterized by UV-visible, mass, and proton NMR spectral analyses and identified as 7-Cl-BA trans-3,4-, 5,6-, and 8,9-dihydrodiols, 3-hydroxy-7-Cl-BA, and 4-hydroxy-7-Cl-BA. Proton NMR spectral analysis indicated that 7-Cl-BA trans-3,4-dihydrodiol preferentially adopted a quasidiequatorial conformation while trans-5,6- and 8,9-dihydrodiols preferentially adopted a quasidiaxial conformation. Comparison of circular dichroism spectra with those of 7-bromobenz(a)anthracene trans-dihydrodiol metabolites of known absolute stereochemistry indicated that the major enantiomeric 7-Cl-BA trans-3,4-, 5,6-, and 8,9-dihydrodiols had R,R absolute configuration. Application of chiral stationary phase HPLC for direct resolution of the trans-dihydrodiols and their hydrogenated and dechlorinated derivatives enabled determination of the optical purity of each dihydrodiol metabolite obtained from the three microsomal systems. In vitro incubation of 7-Cl-BA under molecular oxygen-18 produced 7-Cl-BA trans-3,4-, 5,6-, and 8,9-dihydrodiols, each containing one oxygen-18 atom. Mass spectral analysis of the dehydration products of the oxygen-18-containing trans-dihydrodiol metabolites indicated that 7-Cl-BA 3S,4R-epoxide and 7-Cl-BA 8R,9S-epoxide were the predominant enantiomeric intermediates.