Abstract A single administration of enantiomerically pure 11,12-dihydrodiols of dibenzo[ a,l]pyrene (DB[ a,l]P) on the back of NMRI mice and subsequent chronic treatment with 12- O-tetradecanoylphorbol 13-acetate (TPA) (initiation/promotion assay) revealed strikingly different carcinogenic activities of both enantiomers. Tumor-initiating activity of (−)-(11 R,12 R)-DB[ a,l]P-dihydrodiol, which is the metabolic precursor of the (−)- anti-(11 R,12 S)-dihydrodiol (13 S,14 R)-epoxide, was exceptionally higher than the corresponding effect of (+)-(11 S,12 S)-DB[ a,l]P-dihydrodiol, the metabolic precursor of (+)- syn-(11 S,12 R)-dihydrodiol (13 S,14 R)-epoxide. After topical application of 10 nmol (−)-11,12-dihydrodiol and promotion with TPA twice weekly for a further 18 weeks 93% of treated animals exhibited four to five tumors. In contrast, no neoplasms were observed after treatment with 10 nmol (+)-11,12-dihydrodiol, whereas in the group exposed to 20 nmol of this enantiomer only 13% of mice developed neoplasms (0.1 tumors/survivor). For DB[ a,l]P, considered as the most potent carcinogenic polycyclic aromatic hydrocarbon to date, stereoselective formation of (+)- syn- and (−)- anti-11,12-dihydrodiol 13,14-epoxides via the corresponding enantiomeric 11,12-dihydrodiols has been found to be the principal metabolic activation pathway leading to DNA adducts and mutagenicity. Our study demonstrates that the striking difference in carcinogenic activity in mouse skin of (+)-(11 S,12 S)- and (−)-(11 R,12 R)-DB[ a,l]P-dihydrodiol convincingly reflects the different genotoxicity, i.e. DNA binding and mutagenicity, of both enantiomers observed earlier.