The DNA-dependent protein kinase catalytic subunit (DNA-PK_CS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PK_CS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PK_CS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PK_CS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PK_CS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PK_CS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PK_CS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PK_CS with the DNA ends.