Human DNA polymerase iota is a lesion bypass polymerase of the Y family, capable of incorporating nucleotides opposite a variety of lesions. With undamaged templating purines polymerase iota normally favors Hoogsteen base pairing with syn template. This unusual base pairing is well suited to insertion opposite guanine minor groove lesions, placing the lesion on the spacious major groove side of the polymerase. However, counterintuitively, certain major groove lesions are also bypassed by poli. We have used molecular modeling and dynamics simulations to investigate this phenomenon. Primer extension studies have shown that poli is capable of near-error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetyl-aminofluorene (dG-AAF). Our results suggest that normal Watson-Crick base pairing with template anti could be employed in poli for bypass of dG-AAF. In poli with Hoogsteen paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Futhermore, polymerase iota can incorporate nucleotides opposite a 10S-(+)-trans-anti -[BP]-N⁶-dA lesion (dA*); while mainly error-free, the identity of misincorporated bases is influenced by local sequence context. Our results suggest that hydrogen bonds between the benzo[ a]pyrenyl moiety and nearby bases encourage the templating base to maintain the anti glycosidic bond conformation in the binary complex in a 5'-CAGA*TT-3' sequence. This facilitates correct incorporation of dT via a Watson-Crick pair. In a 5'-TTTA*GA-3' sequence the lesion does not form these hydrogen bonds, permitting dA* to rotate around the glycosidic bond to syn and incorporate dT via a Hoogsteen pair. With syn dA* there is also an opportunity for increased misincorporation of dGTP. Together these results expand our understanding of polymerase iota's lesion bypass capabilities. Specifically, our results suggest that polymerase iota can bypass not only minor groove lesions utilizing Hoogsteen base pairing but also major groove lesions utilizing Watson-Crick base pairing.