Adducts derived from the covalent binding of two positional monomethyl-substituted isomers of a bay region chrysene diol epoxide to supercoiled pIBI30 DNA (2926 base pairs/genome) were prepared, and their characteristics were investigated by a combination of gel electrophoresis and flow linear dichroism techniques. The 5- and 6-methyl derivatives of trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene [(+)-5- and (+)-6-MeCDE, respectively], both with 1R,2S,3S,4R stereochemistry, are characterized by significant differences in their biological activities [Melikian et al. (1988) Cancer Res. 48, 1781–1787]. When covalently bound to plasmid DNA, these two molecules give rise to striking differences in the gel electrophoretic and flow hydrodynamic characteristics of the modified supercoiled DNA. The hydrodynamic flow linear dichroism of linearized DNA molecules (obtained by EcoRI enzyme digestion of covalently closed supercoiled pIBI30 DNA), modified covalently with the highly tumorigenic and mutagenic (+)-5-MeCDE derivative, indicates that flexible joints, bends, or kinks are formed at the site of binding of (+)-5-MeCDE. Slab gel data, as well as ethidium bromide-titration tube agarose gel electrophoresis data, indicate that the formation of (+)-5-MeCDEDNA lesions causes the removal of super helical turns with an unwinding angle of 13 ± 3° per covalently bound polycyclic aromatic residue. In contrast, the biological inactive (+)-6-MeCDE does not significantly alter the characteristics of supercoiled DNA, the unwinding angle is only 2.7 ± 1°, and the changes in persistence lengths detected by the flow linear dichroism technique are significantly smaller than in the case of (+)-5-MeCDEDNA adducts. The observed differences in unwinding effects and alterations in persistence lengths induced by the covalent binding of (+)-5-MeCDE and the (+)-6-MeCDE isomer parallel those observed with DNA adducts derived from the covalent binding of the highly active (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene [(+)-BPDE] isomer and its less active (−)-BPDE enantiomer to øX174 supercoiled DNA [Xu et al. (1992) Nucleic Acids Res. 20, 6167–6176].