DNA-dependent protein kinase (DNA-PK) is required for the repair of double-stranded DNA breaks through the nonhomologous DNA end joining pathway. DNA-PK activity is required for DNA repair, but kinase activity also appears to be attenuated through an autoregulatory feedback loop. We show that autophosphorylation of DNA-PK catalytic subunit occurs in trans at least three sites NH(2) terminal to the catalytic domain and that two sites, threonine 2638 and 2647, determine DNA-PK autophosphorylation in vitro. Thr2638/2647ala substitution in DNA-PK catalytic subunit compromised cellular resistance to ionizing radiation without affecting DNA end joining, suggesting a requirement for DNA-PK inactivation for cell survival at a step after the rejoining of double-stranded DNA breaks.