Double-strand break (DSB) repair in mammalian cells involves the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of three subunits, a catalytic kinase subunit (DNA-PKcs) mutated in the mouse scid complementation group and the Ku heterodimer. Ku-deficient and DNA-PKcs-deficient cell lines and an animal model have illuminated important features of the DSB repair pathway. Additional factors relevant to several essential functions of DNA replication may point to some overlap between DNA synthesis and DNA repair in eukaryotes. The DNA-PK complex may have broader functions based on its possible involvement in transcriptional regulation and cell cycle checkpoints.