Poly(ADP-ribose) polymerase (PARP) senses DNA breaks and facilitates DNA repair via the polyADP-ribosylation of various DNA binding and repair proteins. We explored the mechanism of potentiation of temozolomide cytotoxicity by the PARP inhibitor ABT-888. We showed that cells treated with temozolomide need to be exposed to ABT-888 for at least 17 to 24 hours to achieve maximal cytotoxicity. The extent of cytotoxicity correlates with the level of double-stranded DNA breaks as indicated by gammaH2AX levels. In synchronized cells, damaging DNA with temozolomide in the presence of ABT-888 during the S phase generated high levels of double-stranded breaks, presumably because the single-stranded DNA breaks resulting from the cleavage of the methylated nucleotides were converted into double-stranded breaks through DNA replication. As a result, treatment of temozolomide and ABT-888 during the S phase leads to higher levels of cytotoxicity. ABT-888 inhibits poly(ADP-ribose) formation in vivo and enhances tumor growth inhibition by temozolomide in multiple models. ABT-888 is well tolerated in animal models. ABT-888 is currently in clinical trials in combination with temozolomide.