The DNA-dependent protein kinase (DNA-PK) phosphorylates Sp1 and several other nuclear proteins. Here, we show that Sp1 and the DNA-PK must be colocalized on the same DNA molecule for efficient phosphorylation to occur. Interestingly, we find that the DNA-PK binds to and is activated by the ends of DNA molecules. Furthermore, we show that the DNA binding properties of the DNA-PK are identical to those of Ku, a well-characterized human autoimmune antigen. We demonstrate that the DNA-PK can be fractionated into two components, one of which is Ku and the other of which is a polypeptide of approximately 350 kd. DNA cross-linking and coimmunoprecipitation studies indicate that the catalytic 350 kd DNA-PK component is directed to DNA by protein-protein interactions with Ku. The implications of the unusual DNA binding mode and multicomponent nature of the DNA-PK are discussed.