This paper reports structural characterization of the adducts and tetraols formed from syn-benzo[ghi]fluoranthene-3,4-dihydrodiol-5,5a-epoxide (syn-B[ghi]FDE, 3) and comparative DNA-binding and mutagenicity studies involving 3, anti-B[ghi]FDE (2), and anti-benzo[c]phenanthrene-11,12-dihydrodiol-13,14-epoxide (anti-BcPDE, 5). The structures of nine DNA adducts and two racemic tetraols derived from 3 have been determined spectroscopically. Similar characterization of adducts obtained from the anti-isomer 2 was described in the preceding paper in this issue [Chang et al. (2002) Chem. Res. Toxicol. 15, 187-197]. The majority of DNA adducts with 3 are those from the trans- or cis-opening of the epoxide at C5a by the exocyclic amino groups of dG, dA, and dC. The diolepoxides 2 and 3 are rigid structure analogues of anti- and syn-BcPDE (5 and 6), respectively, thus serving as models for probing molecular deformity and diol conformation in diolepoxide-DNA interaction. Comparative DNA binding experiments indicate that 57% of 2 and 33% of 3 were converted into DNA adducts, whereas a 71% conversion was observed for 5. In general, lower percentages were observed with denatured calf-thymus DNA. As for base selectivity, 2 showed a greater affinity for dA relative to dG (dA/dG ratio, 0.79) than 3 (0.56) when reacted with native calf-thymus DNA. A much higher dA/dG ratio (1.41) was obtained for 5. The overall dA/dG ratios were lower with denatured DNA, indicating the importance of the secondary structure of DNA for both adduct formation and chemical selectivity. The T-shape pseudo-diaxial diols of 3 appears to have favorable electrostatic interactions with the nearby phosphate backbone in the minor groove of DNA, thereby yielding greater amounts of dG adducts than the pseudo-diequatorial 2. The anti-isomer 2 was found to be seven times more mutagenic than 3, but they are significantly less mutagenic than the nonplanar analogue 5 when tested in Salmonella typhimurium TA 100.