The DNA-dependent protein kinase (DNA-PK) requires for activity free ends or other discontinuities in the structure of double strand DNA. In vitro, DNA-PK phosphorylates several transcription factors and other DNA-binding proteins and is thought to function in DNA damage recognition or repair and/or transcription. Here we show that in vitro DNA-PK undergoes autophosphorylation of all three protein subunits (DNA-PKcs, Ku p70 and Ku p80) and that phosphorylation correlates with inactivation of the serine/threonine kinase activity of DNA-PK. Significantly, activity is restored by the addition of purified native DNA-PKcs but not Ku, suggesting that inactivation is due to autophosphorylation of DNA-PKcs. Our data also suggest that autophosphorylation results in dissociation of DNA-PKcs from the Ku-DNA complex. We suggest that autophosphorylation is an important mechanism for the regulation of DNA-PK activity.