Human DNA polymerase iota (pol(iota)) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that poliota is able to facilitate limited translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that pol(iota) misinserts T or G opposite the 3' T of the CPD approximately 1.5 times more frequently than the correct base, A. While pol(iota) is unable to extend the T.T mispair significantly, the G.T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A. T base pair. By comparison, pol(iota) readily misinserts two bases opposite a 6-4 thymine-thymine pyrimidine-pyrimidone photoproduct (6-4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that poliota possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.