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This study demonstrates that benzo[ g]chrysene-11,12-dihydrodiol (B[ g]C-11,12-dihydrodiol) derived from the fjord-region parent hydrocarbon B[ g]C is oxidized by rat AKR1C9 with a kcat/ Km100 times greater than that observed with the commonly studied bay-region benzo[ a]pyrene-7,8-dihydrodiol (B[ a]P-7,8-dihydrodiol). Conversely, despite its strikingly similar structure to B[ g]C-11,12-dihydrodiol, benzo[ c]phenanthrene-3,4-dihydrodiol (B[ c]Ph-3,4-dihydrodiol) is consumed by AKR1C9 at sluggish rates comparable to those observed with B[ a]P-7,8-dihydrodiol. CD spectroscopy revealed that only the (+)-B[ g]C-11,12-dihydrodiol stereoisomer was oxidized, while AKR1C9 oxidized both stereoisomers of B[ a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol. The (+)- S, S- and (−)- R, R-stereoisomers of B[ g]C-11,12-dihydrodiol were purified by chiral RP-HPLC. The 11 S,12 S-stereoisomer was oxidized at the same rate as the racemate. The 11 R,12 R-stereoisomer did not act as an inhibitor to AKR1C9, indicating that the (−)- R, R-stereoisomer was excluded from the active site. To understand the basis of stereochemical preference, we screened alanine-scanning mutants of active site residues of AKR1C9. These studies revealed that in comparison to the wild type, F129A, W227A, and Y310A enabled the oxidation of both the B[ g]C-11 S,12 S-dihydrodiol and the B[ g]C-11 R,12 R-dihydrodiol. Molecular modeling revealed that unlike B[ a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol, B[ g]C-11,12-dihydrodiol enantiomers are significantly bent out of plane. As a consequence, the (−)- R, R-stereoisomer was prevented from binding to the active site because of unfavorable interactions with F129, W227, or Y310. Additionally, LC/MS validated that the product of the reaction of B[ g]C-11,12-dihydrodiol oxidation catalyzed by AKR1C9 was B[ g]C-11,12-dione, which was trapped in vitrowith the nucleophile 2-mercaptoethanol. The similarity between rates of trans-dihydrodiol oxidation by the rat and human liver specific AKRs (AKR1C9 and AKR1C4) implicate these enzymes in hepatocarcinogenesis in rats observed with the fjord-region PAH. [ABSTRACT FROM AUTHOR]