Thermal radiosensitization is thought to result from inhibition of repair of radiation-induced DNA damage, DNA double-strand breaks in particular. Since the DNA-dependent protein kinase (DNA-PK) complex plays a major role in the nonhomologous end-joining of DSBs, it has been suggested that inactivation of this complex as a whole or of its individual subunits by heat might be involved in radiosensitization by heat. To test this hypothesis further, the ability of heat to enhance the radiosensitivity of cells proficient or deficient in either Ku80 or the DNA-PK catalytic subunit (DNA-PKcs) was investigated. In cells of two Ku80-deficient and two DNA-PKcs-deficient and double-strand break-deficient cell lines, the extent of radiosensitization by heat was not reduced compared to that in both their isogenic gene-complemented counterparts as well as to that in their parental cells. Thus radiosensitization by hyperthermia can be obtained irrespective of the Ku80 or DNA-PKcs status in cells. Therefore, Ku80 or DNA-PKcs and hence nonhomologous DSB end-joining do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.