Three proteins known to play a critical role in mammalian DNA double- strand break repair and lymphoid V(D)J recombination are the autoantigens Ku86 and Ku70 and a 465-kDa serine/threonine protein kinase catalytic subunit (DNA-PK(Ca)). These proteins physically associate to form a complex (DNA·PK) with DNA-dependent protein kinase activity. In this study, we demonstrate using electrophoretic mobility shift assays (EMSAs) that the nuclear DNA end- binding activity of Ku is altered in the human promyelocytic leukemic HL-60 cell line. Western blot and EMSA supershift analyses revealed that HL-60 cells expressed both full-length and variant Ku86 proteins. However, a combined EMSA and immunoanalysis revealed that the Ku heterodimers complexed with DNA in HL-60 cells contained only the variant Ku86 proteins. Finally, UV cross-linking experiments and DNA·PK assays demonstrated that the Ku complexes containing variant Ku86 had a greatly reduced ability to interact with DNA-PK(Ca) and that consequently HL-60 cells had severely diminished DNA·PK activity. These data provide important insights into the interaction between Ku and DNA-PK(Ca) and into the role of DNA·PK in DNA double-strand break repair.