AG14361 is a potent PARP-1 inhibitor, with a Ki of < 5 nM, and in permeabilized SW620 and intact SW620 cells, the IC50s are 29 nM and 14 nM, respectively. AG14361 inhibits the proliferation of human cancer cells, such as A549, LoVo, and SW620 cells, with GI50s of 14 μM, 11.2 μM and 20 μM, respectively. Furthermore, AG14361 in combination with NSC 362856 markedly reduces the GI50 value of NSC 362856 in LoVo and A549 cells, but does not exert such an effect in SW620 cells. AG14361 suppresses breast cancer cells with IC50s of 17 μM and 25 μM for 92 J-wt-BRCA1 and 92 J-sh-BRCA1 cells, respectively. AG14361 induces caspase 3/7 activation and cell cycle abnormalities, and also inhibits NF-κB signaling. AG14361 (0.4 μM) enhances the growth-inhibitory and cytotoxic effects of topoisomerase I poisons, with no obvious effect on the formation and reversal of cleavable complexes, and increases the persistence of camptothecin-induced DNA single-strand breaks.