We demonstrate that the catalytic subunit of the DNA-dependent protein kinase (DNA-PK(cs)) is specifically, proteolytically cleaved in HL-60 cells treated with staurosporine (STS), a potent inducer of apoptosis. The proteolysis of DNA-PK(cs) correlated with or preceded apoptotic chromosomal DNA degradation. Cell-free extracts prepared from STS-treated HL-60 cells recapitulated the proteolysis of DNA-PK(cs) in an in vitro assay using purified DNA-PK as the substrate. Western blot analyses of the apoptotic cell extract showed that the 32-kDa precursor of CPP32 is expressed in HL-60 cells and processed following STS treatment. In addition, whereas the DNA-PK(CS) protease activity was not inhibitable by many conventional protease inhibitors, it was inhibitable by a highly selective peptide-derived inhibitor of CPP32. These data strongly suggest that CPP32, or a CPP32-like protease, is responsible for DNA-PK(CS) proteolysis. Finally, our results demonstrated that the cleavage of DNA-PK(cs) in vitro proceeded in the presence of Bcl-2, indicating that the function provided by Bcl-2 lies upstream the proteolysis of DNA-PK(cs).