DNA lesions that escape excision repair pathways can cause arrested DNA replication. This replication block can be processed by translesion DNA synthesis (TLS), which is carried out by a number of specialized DNA polymerases. A sequential lesion bypass model has been proposed; one of the lesion-specific polymerases inserts nucleotide(s) opposite the damaged template, followed by extension from the inserted nucleotide by the same or another polymerase. Polzeta and Polkappa have been proposed as candidates for executing the extension step in eukaryotic cells. We previously disrupted separately Rev3, the catalytic subunit of Polzeta, and Polkappa in chicken B lymphocyte DT40 cells. We found that each cell line showed significant UV sensitivity, implying that both contribute to UV radiation damage repair. In the present studies we generated REV3(-/-)POLK(/-) double knock-out cells to determine whether they participate in the same or different pathways. The double mutant was viable and proliferated with the same kinetics as parental REV3(-/-) cells. The cells showed the same sensitivity as REV3(-/-) cells to UV, ionizing radiation, and chemical cross-linking agents. In contrast, they were more sensitive than REV3(-/-) cells to monofunctional alkylating agents, even though POLK(/-) cells barely exhibited increased sensitivity to those. Moreover Polk-deficient mouse embryonic stem and fibroblast cells, both of which have previously been shown to be sensitive to UV radiation, also showed moderate sensitivity to methyl methanesulfonate, a monofunctional alkylating agent. These data imply that Polkappa has a function in TLS past alkylated base adducts as well as UV radiation DNA damage in vertebrates.