Radiosensitive cell lines derived from X‐ray cross complementing group 5 (XRCC5), SCID mice and a human glioma cell line lack components of the DNA‐dependent protein kinase, DNA‐PK, suggesting that DNA‐PK plays an important role in DNA double‐strand break repair. Another enzyme implicated in DNA repair, poly(ADP‐ribose) polymerase, is cleaved and inactivated during apoptosis, suggesting that some DNA repair proteins may be selectively targeted for destruction during apoptosis. Here we demonstrate that DNA‐PKcs, the catalytic subunit of DNA‐PK, is preferentially degraded after the exposure of different cell types to a variety of agents known to cause apoptosis. However, Ku, the DNA‐binding component of the enzyme, remains intact. Degradation of DNA‐PKcs was accompanied by loss of DNA‐PK activity. One cell line resistant to etoposide‐induced apoptosis failed to show degradation of DNA‐PKcs. Protease inhibitor data implicated an ICE‐like protease in the cleavage of DNA‐PKcs, and it was subsequently shown that the cysteine protease CPP32, but not Mch2alpha, ICE or TX, cleaved purified DNA‐PKcs into three fragments of comparable size with those observed in cells undergoing apoptosis. Cleavage sites in DNA‐PKcs, determined by antibody mapping and microsequencing, were shown to be the same for CPP32 cleavage and for cleavage catalyzed by extracts from cells undergoing apoptosis. These observations suggest that DNA‐PKcs is a critical target for proteolysis by an ICE‐like protease during apoptosis.