The present study investigated the relationship between DNA-dependent protein kinase (DNA-PK) and radiosensitivity of nasopharyngeal carcinoma (NPC) cell lines. The dose-survival relationship for NPC cell lines, CNE1 and CNE2, was analyzed using clonogenic formation assay, the activity of DNA-PK of the two cell lines was measured using the Signa TECT DNA-PK assay kit, and the localization and expression of Kus (a heterodimer) and DNA-PKcs protein in CNE1 and CNE2 before irradiation and 15 min, 1 h, 6 h, 12 h, 24 h after 4 Gy irradiation were analyzed by immunofluorescence, laser scanning confocal microscope (LSCM) and Western blot. The results showed that the surviving fraction of CNE1 was higher than that of CNE2 at each dose. The DNA-PK activity of CNE1 was also significantly higher than that of CNE2 before and after irradiation (P<0.05), while the expression of total Ku70/Ku80 in CNE1 and CNE2 had no significant difference. Increasing translocation of Ku70 and Ku80 from the cytoplasm to the nuclei in the two cell lines was observed with increase of irradiation time as detected by Western blot, and the immunofluorescence of the DNA-PK complex subunits showed greater nuclear translocation in CNE1 than CNE2 after irradiation. The results suggest that the relatively higher radio-resistance of CNE1 correlates with the higher activity of DNA-PK as compared to that of more radiosensitive CNE2 (or lower radio-resistance) before and after irradiation. Thus, DNA-PK activity may be a useful predictor of radiosensitivity of NPC.