The major cytotoxic DNA adduct induced by temozolomide and other methylating agents used in malignant glioma and metastasized melanoma therapy is O(6)-methylguanine (O(6)-MeG). This primary DNA damage is converted by mismatch repair into secondary lesions, which block replication and in turn induce DNA double-strand breaks that trigger the DNA damage response (DDR). Key upstream players in the DDR are the phosphoinositide 3-kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR). Here, we addressed the question of the importance of ATM and ATR in the cell death response following temozolomide. We show that (i) ATM- and ATR-mutated cells are hypersensitive to temozolomide, (ii) O(6)-MeG triggers ATM and ATR activation, (iii) knockdown of ATM and ATR enhances cell kill in gliobalstoma and malignant melanoma cells with a stronger and significant effect in ATR knockdown cells, (iv) ATR, but not ATM, knockdown abolished phosphorylation of H2AX, CHK1, and CHK2 in glioma cells, and (v) temozolomide-induced cell death was more prominently enhanced by pharmacologic inhibition of CHK1 compared with CHK2. The data suggest that ATM and, even better, ATR inhibition is a useful strategy in sensitizing cancer cells to temozolomide and presumably also other anticancer drugs.