Human DNA polymerase iota (Poliota) is a member of the Y family of DNA polymerases involved in translesion DNA synthesis. Poliota is highly unusual in that it possesses a high fidelity on template A, but has an unprecedented low fidelity on template T, preferring to misincorporate a G instead of an A. To understand the mechanisms of nucleotide incorporation opposite different template bases by Poliota, we have carried out pre-steady-state kinetic analyses of nucleotide incorporation opposite templates A and T. These analyses have revealed that opposite template A, the correct nucleotide is preferred because it is bound tighter and is incorporated faster than the incorrect nucleotides. Opposite template T, however, the correct and incorrect nucleotides are incorporated at very similar rates, and interestingly, the greater efficiency of G misincorporation relative to A incorporation opposite T arises predominantly from the tighter binding of G. Based on these results, we propose that the incipient base pair is accommodated differently in the active site of Poliota dependent upon the template base and that when T is the templating base, Poliota accommodates the wobble base pair better than the Watson-Crick base pair.