DNA-dependent protein kinase (DNA-PK) and poly (ADP-ribose) polymerase-1 (PARP-1) participate in nonhomologous end joining and base excision repair, respectively, and are key determinants of radio- and chemo-resistance. Both PARP-1 and DNA-PK have been identified as therapeutic targets for anticancer drug development. Here we investigate the effects of specific inhibitors on enzyme activities and DNA double-strand break (DSB) repair. The enzyme activities were investigated using purified enzymes and in permeabilized cells. Inhibition, or loss of activity, was compared using potent inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361), and cell lines proficient or deficient for DNA-PK or PARP-1. Inactive DNA-PK suppressed the activity of PARP-1 and vice versa. This was not the consequence of simple substrate competition, since DNA ends were provided in excess. The inhibitory effect of DNA-PK on PARP activity was confirmed in permeabilized cells. Both inhibitors prevented ionizing radiation-induced DSB repair, but only AG14361 prevented single-strand break repair. An increase in DSB levels caused by inhibition of PARP-1 was shown to be caused by a decrease in DSB repair, and not by the formation of additional DSBs. These data point to combined inhibition of PARP-1 and DNA-PK as a powerful strategy for tumor radiosensitization.