In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.