Dibenzo[a,l]pyrene (DBP) is one of the most potent bacterial mutagen and mammary carcinogens. When DBP (50 microM) was incubated with calf thymus DNA (300 microg/ml) in the presence of liver microsomes from beta-naphthoflavone (beta-NF)- or Aroclor 1254-treated rats, at least eight adduct spots were detected as analyzed by nuclease P1-enhanced 32P-postlabeling assay. DNA adduction was enhanced by nearly 20- and 60-fold with beta-NF- and Aroclor 1254-induced microsomes, respectively, as compared with uninduced microsomes, suggesting a possible involvement of CYP1A family in DBP activation. Inclusion of the selective P4501A1 inhibitor, alpha-naphthoflavone (50 microM) in the activation reaction almost completely (>98%) abolished adduct formation further supporting involvement of P4501A in DBP activation. Analysis of DNA and 2'-deoxynucleosides 3'-mononucleotide reacted with anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDEs) and co-chromatography analyses in multiple solvents showed that the microsomal DBP-DNA adducts were derived by interaction of both anti- and syn-DBPDEs with adenine and guanine in DNA in the following order: anti-DBPDE-dA approximately syn-DBPDE-dG >> anti-DBPDE-dG approximately syn-DBPDE-dA. It is concluded that (i) most or all DBP adducts were P4501A-mediated; (ii) both the anti- and syn-stereoisomers were involved in the DNA adduct formation; and (iii) both adenine and guanine in the DNA contributed equally to the formation of the major and minor adducts.