The DNA-dependent protein kinase (DNA-PK) complex is a serine/threonine protein kinase comprised of a 469-kDa catalytic subunit (DNA-PK_cs) and the DNA binding regulatory heterodimeric (Ku70/Ku86) complex Ku. DNA-PK functions in the nonhomologous end-joining pathway for the repair of DNA double-stranded breaks (DSBs) introduced by either exogenous DNA damage or endogenous processes, such as lymphoid V(D)J recombination. Not surprisingly, mutations in Ku70, Ku86, or DNA-PK_cs result in animals that are sensitive to agents that cause DSBs and that are also immune deficient. While these phenotypes have been validated in several model systems, an extension of them to humans has been missing due to the lack of patients with mutations in any one of the three DNA-PK subunits. The worldwide lack of patients suggests that during mammalian evolution this complex has become uniquely essential in primates. This hypothesis was substantiated by the demonstration that functional inactivation of either Ku70 or Ku86 in human somatic cell lines is lethal. Here we report on the functional inactivation of DNA-PK_cs in human somatic cells. Surprisingly, DNA-PK_cs does not appear to be essential, although the cell line lacking this gene has profound proliferation and genomic stability deficits not observed for other mammalian systems.