How DNA damage is converted into intracellular signals that can control cell behaviour is unknown. The c-Abl protein tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of a serine/threonine kinase and Ku DNA-binding subunits, requires DNA double-strand breaks or other DNA lesions for activation. Here we demonstrate that c-Abl interacts constitutively with DNA-PK. Ionizing radiation stimulates binding of c-Abl to DNA-PK and induces an association of c-Abl with Ku antigen. We show that DNA-PK phosphorylates and activates c-Abl in vitro. Cells deficient in DNA-PK are defective in c-Abl activation induced by ionizing radiation. In a potential feedback mechanism, c-Abl phosphorylates DNA-PK, but not Ku, in vitro. Phosphorylation of DNA-PK by c-Abl inhibits the ability of DNA-PK to form a complex with DNA. We also show that treatment of cells with ionizing radiation results in phosphorylation of DNA-PK that is dependent on c-Abl. Our results support the hypothesis that there are functional interactions between c-Abl and DNA-PK in the response to DNA damage.