The DNA repair enzymes, DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1), are key determinants of radio- and chemo-resistance. We have developed and evaluated novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) for use in anticancer therapy. PARP-1- and DNA-PK-deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR) in G₀ cells, compared with their proficient counterparts. NU7026 (10 μm) potentiated IR cytotoxicity [potentiation factor at 90% cell kill (PF₉₀) = 1.51 ± 0.04] in exponentially growing DNA-PK proficient but not deficient cells. Similarly, AG14361 (0.4 μm) potentiated IR in PARP-1^+/+ (PF₉₀ = 1.37 ± 0.03) but not PARP-1^−/− cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive (e.g., PF₉₀ = 2.81 ± 0.19 in PARP-1^+/+ cells). Both inhibitors alone reduced PLDR ∼3-fold in the proficient cell lines. Furthermore, the inhibitor combination completely abolished PLDR. IR-induced DNA double strand break (DNA DSB) repair was inhibited by both NU7026 and AG14361, and use of the inhibitor combination prevented 90% of DNA DSB rejoining, even 24-h postirradiation. Thus, there was a correlation between the ability of the inhibitors to prevent IR-induced DNA DSB repair and their ability to potentiate cytotoxicity. Thus, individually, or in combination, the DNA-PK and PARP-1 inhibitors act as potent radiosensitizers and show potential as tools for anticancer therapeutic intervention.