The phosphatidylinositol 3-kinase/AKT pathway is activated frequently in human cancer, and it has been implicated in tumor cell proliferation, survival, and chemoresistance. In this study, we addressed the role of AKT in cellular responses to the therapeutic methylating agent temozolomide (TMZ), and we investigated the possible link between TMZ-induced modulation of AKT function and activation of ataxia-telangiectasia and Rad3-related (ATR)- and ataxia telangiectasia mutated (ATM)-dependent signaling pathways. We found that clinically relevant concentrations of TMZ caused activation of endogenous AKT in lymphoblastoid cells, and in colon and breast cancer cells, and that this molecular event required a functional mismatch repair system. Transfection of a dominant-negative kinase-dead form of AKT1 into breast cancer cells abrogated TMZ-induced activation of endogenous AKT, and it markedly enhanced cell sensitivity to the drug. Likewise, exposure of the MMR-proficient cell lines to the AKT inhibitor D-3-deoxy-2-O-methyl-myo inositol 1-[(R)-2-methoxy-3-(octadecyloxy)-propyl hydrogen phosphate] (SH-5) impaired AKT phosphorylation in response to TMZ, and it significantly increased cell chemosensitivity. Furthermore, small interfering RNA (siRNA)-mediated reduction of AKT1 expression in colon cancer cells potentiated the growth inhibitory effects of TMZ. Inhibition of ATM expression in colon cancer cells by siRNA did not impair TMZ-induced activation of AKT, whereas siRNA-mediated inhibition of ATR prevented AKT activation in response to the drug and increased cell chemosensitivity. These results strongly support the hypothesis that clinical benefit could be obtained by combining TMZ with inhibitors of the AKT pathway. Moreover, they provide the first evidence of a novel function of ATR as an upstream activator of AKT in response to DNA damage induced by O(6)-guanine-methylating agents.