Ku plays an important role in multiple nuclear processes, e.g., DNA repair, chromosome maintenance, and transcriptional regulation. Although some evidence suggests that the nuclear translocation of Hu plays a key role in regulating the function of Ku, the mechanism is poorly understood. Using the site-directed mutagenesis technique, we demonstrate here that Ku70 can translocate to the nucleus without heterodimerization with Ku80. The nuclear accumulation of Ku70 mutants of the nuclear localization signal, which retained their binding ability with Ku80, was diminished. On the other hand, Ku70 mutants which lacked the ability to bind with Ku80 could translocate to the nuclei. Human Ku70, when transfected, accumulated within the nuclei of hamster xrs-6 cells which had undetectable DNA-PK activity and Ku80. Ku70 and Ku80 mutants of DNA-PK phosphorylation sites showed normal heterodimerization and nuclear translocation. These findings also support the idea that Ku70 can translocate to the nucleus independent of DNA-PK autophosphorylation.