O(6) -Methylguanine (O(6) -MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O(6) -MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O(6) -MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O(6) -MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O(6) -MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O(6) -MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O(6) -MeG without a significant loss of efficiency.