We have constructed a series of polyomavirus-based shuttle vectors for analyzing mechanisms of mutagenesis in rodent cell systems. These vectors contain the supF suppressor tRNA gene which serves as the mutagenesis target; the pBR327 replication functions and ampr gene for replication and selection in bacteria; and the polyomavirus genome which permits replication in rodent cells. The polyoma genomes used in these vectors vary in their enhancer regions, causing varying efficiencies of replication in different types of rodent cells. One of the vectors (pPySLPT-2) which replicates particularly well in several different rodent cell types (i.e., Chinese hamster ovary, mouse hepatoma and mouse lymphoma) was used to compare mutation induction by UV radiation in UV repair-deficient mouse lymphoma L5178Y-R cells with mutagenesis in the related UV repair-proficient line, L5178Y-S. In both cell types, UV-induced mutants could be recovered at frequencies up to 50-fold higher than that of the spontaneous background. At a given UV fluence the L5178Y-R cells were more highly mutable than the L5178Y-S cells. Our results indicate that these new polyomavirus-based vectors should be useful for analysis of the molecular mechanisms of mutation induction in rodent cell systems, and in particular should allow detailed analysis of mutagenesis in the well characterized rodent somatic cell mutants.