Mammalian DNA polymerase μ (pol μ) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol μ protein increase. pol μ also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of γH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol μ is thus part of the cellular response to DNA double-strand breaks. pol μ also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol μ in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase β does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol μ in facilitating joining mediated by these factors. Our data thus support an important role for pol μ in the end-joining pathway for repair of double-strand breaks.