DNA damage, which is left unrepaired by excision repair pathways, often blocks replication, leading to lesions such as breaks and gaps on the sister chromatids. These lesions may be processed by either homologous recombination (HR) repair or translesion DNA syntbesis (TLS). Vertebrate Polκ belongs to the DNA polymerase Y family, as do most TLS polymerases. However, the role for Polκ in vertebrate cells is unclear because of the lack of reverse genetic studies. Here, we generated cells deficient in Polκ (polκ cells) from the chicken B lymphocyte line DT40. Although purified Polκ is unable to by-pass ultraviolet (UV) damage, polκ cells exhibited increased UV sensitivity, and the phenotype was suppressed by expression of human and chicken Polκ, suggesting that Polκ is involved in TLS of UV photoproduct. Defects in both Polκ and Rad18, which regulates TLS in yeast, in DT40 showed an additive effect on UV sensitivity. Interestingly, the level of sister chromatid exchange, which reflects HR-mediated repair, was elevated in normally cycling polκ cells. This implies functional redundancy between HR and Polκ in maintaining chromosomal DNA. In conclusion, vertebrate Polκ is involved in Rad18-independent TLS of UV damage and plays a role in maintaining genomic stability.