We have recently shown that irradiating cells prior to transfection induces recombination, as manifested by increased stable transduction of both plasmid and adenoviral vectors. We hypothesized that Ku proteins, which have previously been shown to be involved in both recombination and the repair of DNA damage after irradiation, would likely be important mediators of radiation-induced recombination. The present work demonstrates that Ku80 is essential for radiation-induced recombination. While human and hamster Ku80 are equally effective at restoring the transfection efficiency and radiation resistance of xrs-5 cells, human Ku80 is much more effective at radiation-induced recombination than hamster Ku80. This difference is not due to differences in Ku80 expression or DNA end-binding activity, but it may be due to structural differences between human and hamster Ku80.