Previously, we have shown that integrated copies of polyoma DNA can be induced to replicate in rat fibroblasts (H3 cells) exposed to a DNA-damaging agent. In the current study, we demonstrate that UV-irradiation of mouse fibroblasts (WOP cells), transiently transfected with polyoma DNA, results in repression of polyoma replication. Cotransfection of oligomers representing wild-type but not mutated forms of the PEBP2 target sequence restored levels of viral replication indicating a role of PEBP2 binding proteins in mediating this effect. DNA-binding assays revealed that a different subset of complexes was formed with the PEBP2 target sequence when nuclear proteins from sham and UV-irradiated WOP and H3 cells were compared, suggesting that the activities of PEBP2 binding proteins are differentially regulated upon UV-irradiation in these two cell types. The ability of PEBP2 to modulate polyoma replication following UV-irradiation in WOP cells suggests a potential role of PEBP2 proteins in the cellular response to DNA damage.