Previous studies have shown that telomerase facilitates DNA-damage repair and cell survival following stress. It is not clear how telomerase promotes DNA repair, or whether short-term telomerase inhibition, combined with genotoxic stress, can be exploited for cancer therapy. Here, we show that transient inhibition of telomerase activity by the specific inhibitor, GRN163L, increases the cytotoxicity of some, but not all, DNA-damaging agents. Such synergistic inhibition of growth requires the use of DNA-damaging agents that are toxic in the S/G₂ phase of the cell cycle. Notably, inhibition of Ataxia Telangiectasia Mutated (ATM) kinase, together with telomerase inhibition, synergistically increases the cytotoxicity induced by the G₂-specific topoisomerase II inhibitor etoposide. By varying the timing of telomerase inhibition, relative to the timing of DNA damage, it is apparent that the prosurvival functions of telomerase occur at early stages of DNA damage recognition and repair. Our results suggest that the protective role of telomerase in cell cycle–restricted DNA damage repair could be exploited for combined anticancer chemotherapy. Cancer Res; 70(21); 8684–94. ©2010 AACR.