The DNA-dependent protein kinase (DNA-PK) is required for double-strand break repair in mammalian cells. DNA-PK contains the heterodimer Ku and a 460-kDa serine/threonine kinase catalytic subunit (p460). Ku binds in vitro to DNA termini or other discontinuities in the DNA helix and is able to enter the DNA molecule by an ATP-independent process. It is clear from in vitro experiments that Ku stimulates the recruitment to DNA of p460 and activates the kinase activity toward DNA-binding protein substrates in the vicinity. Here, we have examined in human nuclear cell extracts the influence of the kinase catalytic activity on Ku binding to DNA. We demonstrate that, although Ku can enter DNA from free ends in the absence of p460 subunit, the kinase activity is required for Ku translocation along the DNA helix when the whole Ku/p460 assembles on DNA termini. When the kinase activity is impaired, DNA-PK including Ku and p460 is blocked at DNA ends and prevents their processing by either DNA polymerization, degradation, or ligation. The control of Ku entry into DNA by DNA-PK catalytic activity potentially represents an important regulation of DNA transactions at DNA termini.