Two of the enzymes involved in the response of mammalian cells to ionizing radiation are the DNA-dependent protein kinase and poly(ADP-ribose) polymerase. These enzymes are known to be activated by binding to DNA strand breaks, but previous studies designed to look at strand break specificity have employed enzymatically generated strand breaks and not irradiated DNA. Using highly purified DNA-dependent protein kinase, we compared enzyme activation by a series of DNA substrates. Irradiated plasmid DNA activated DNA-dependent protein kinase in a dose-dependent manner. When calculated in terms of the molar concentration of double-strand breaks, the enzyme activation by irradiated DNA was comparable to that by restriction enzyme-cleaved DNA. Linear DNA purified after plasmid irradiation also activated DNA-dependent protein kinase to a comparable extent, but nicked DNA, either isolated from irradiated plasmid or generated by DNase I, failed to activate the enzyme. A comparison of the enzyme activation by plasmid molecules with different 3'- and 5'-terminal groups indicated that the chemical nature of the DNA termini at the double-strand break does not significantly influence the response of the DNA-dependent protein kinase. Similar experiments with poly(ADP-ribose) polymerase demonstrated that single- and double-strand breaks activate this enzyme with almost equal efficiency, but because of their greater number, single-strand breaks dominate the response of poly(ADP-ribose) polymerase to irradiated DNA.