A newly-developed polyomavirus-based shuttle vector, pPySLPT-2, was used to analyze mutations induced by UV radiation in the two related mouse lymphoma cell lines, L5178Y-R (LY-R) and L5178Y-S (LY-S). These well-studied cell lines differ in sensitivity to UV radiation, apparently due to differences in DNA repair capacity. Consistent with these differences, we found that replication of UV-irradiated vector is inhibited to a greater extent in the UV sensitive LY-R cells than in the LY-S cells. Mutations were induced in the vector's supF target gene at a higher frequency in the LY-R cells than in the LY-S cells, but the sequence characteristics of the base substitution mutations were similar in the two cell lines. The pattern of UV-induced mutations in the supF target gene of this polyomavirus-based vector, replicated in the mouse cells, was very similar to the pattern observed in the same target gene in the analogous simian virus 40-based vector, pZ189, replicated in monkey and human cells.