Human DNA polymerase iota (Pol iota) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol iota with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol iota active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N(6) of an A or the O(6) of a G, here we provide evidence that whereas hydrogen bonding at N(6) is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O(6) is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O(6) and N7 hydrogen bonding to DNA synthesis by Pol iota, we have examined its proficiency for replicating through the (6)O-methyl guanine and 8-oxoguanine lesions, which affect the O(6) and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O(6) is an imperative. The dispensability of N(6) hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol iota with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N(6) positions of templating A.