Polyclonal antiserum prepared against DNA that was modified with racemic benzo[c]phenanthrene-3,4-diol-1,2-epoxide-2 (B[c]PhDE-2; benzylic hydroxyl and epoxide oxygen trans) was characterized for specificity of antigen recognition. Previous studies have demonstrated that the antisera stereoselectively recognized B[c]PhDE-2-DNA and failed to recognize DNA modified with racemic benzo[c]phenanthrene-3,4-diol-1,2-epoxide-l (B[c]PhDE-I-DNA, benzylic hydroxyl and epoxide oxygen cis), benzo[a]pyrene-7,8-diol-9,lO-epoxide-2-DNA (B[a]PDE-2-DNA) and 7,12-dimethylbenz-[a]anthracene-3,4-diol-l,2-epoxide-l-DNA (DMBADE-I -DNA). DNA samples modified by diol-epoxide-2 diastereomers of several hydrocarbons were tested in competitive ELISA assays utilizing B[c]PhDE-2-DNA (270 fmol adducts per well). DNA modified with racemic diol-epoxide-2 of various substituted chrysenes (including chrysene, benzok]chrysene (Bk]C), 6-methylchrysene (6-MeC), and 5-methychrysene (5-MeC), gave 50% inhibition of antisera binding at significantly higher concentrations (5 to 7-fold) than the parent B[c]PhDE-2-DNA or 5,6-diMeCDE-DNA. DNA modified with 5,7-dimethylchryseneDE-2 (5,7-diMeCDE-2) and dibenzo[a,l]pyreneDE-2 (DB[a,d PDE-2) required 20 and > 100-fold greater levels of adducts to give 50% inhibition. Results with B[c]Ph, 5,6-diMeC, chrysene, 6-MeC and 5-MeC diol epoxide-2-DNA indicate that substitution of a methyl group in the vicinity of the bay-region of the PAHmolecule had limited effects on antigen recognition by this antiserum. However. the addition of a ring or methyl group remote from the diol epoxide moiety, as in DB[tr.l] PDE-2-DNA or 5,7-diMeCDE-?-DNA greatly decreased antigen recognition. The ability of this antiserum to recognize DNA adducts of a particular class of polycyclic aromatic hydrocarbons will be useful for studies of their contribution to the DNAbinding that results from exposure to complex environmental mixtures.