DNA-dependent protein kinase (DNA-PK) consists of a 460 kDa subunit that contains the catalytic domain (DNA-PKcs) complexed with two polypeptides of 70 kDa and 80 kDa (Ku70 and Ku80) which comprise the Ku autoantigen. DNA-PKcs requires association with DNA via Ku for catalytic activation and is implicated in double strand break repair, V(D)J recombination and transcription. We have utilised a cell-free system of concentrated Xenopus laevis egg extracts to investigate the regulation and possible functions of DNA-PK. Recently, we have shown that this system can reproduce events of apoptosis, including activation of an apoptotic protease that cleaves poly(ADP-ribose) polymerase. Here, we report that DNA-PK is rapidly inactivated with the onset of apoptosis in this system. Loss of activity is concomitant with cleavage of the catalytic subunit, whereas the Ku subunits are stable. Cleavage and inactivation of DNA-PKcs is prevented by prior addition of the anti-apoptotic protein Bcl-2 or inhibition of an apoptotic protease that has characteristics of the CPP-32/Ced-3 family of cysteine proteases that cleave poly(ADP-ribose) polymerase. These results suggest that cleavage and inactivation of DNA-PKcs prevents this factor from functioning in DNA repair, recombination or transcriptional regulation during apoptosis.