The mechanism of radiosensitization by gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is not exactly known. We investigated the possible role of inhibition of the repair of DNA double-strand breaks by dFdC by measuring the extent of radiosensitization in different cell lines deficient and proficient in components of nonhomologous end-joining and in the parental cell lines. Different cell lines were incubated with 0.5 and 5 microM dFdC for 4 h. Cells deficient in DNA-dependent protein-kinase catalytic subunit (V3) showed sensitization similar to that of wild-type cells (AAS) and complemented cells (V3+YAC). Ku80-deficient cells (xrs5 and xrs6) showed even more radiosensitization by dFdC as compared with wild-type CHO-K1. However, Ku80-complemented cell lines (xrs5+huKu80 and xrs6+haKu80) did not show radiosensitization. The differences in dFdC-mediated radiosensitization were not attributable to different changes in deoxynucleotide triphosphate levels and cell cycle distribution. We conclude that a functional nonhomologous end-joining pathway is not required for dFdC-mediated radiosensitization.