Overexpression of N-methylpurine DNA glycosylase (MPG) has been suggested as a possible gene therapy approach to sensitize tumor cells to the cell-killing effects of temozolomide, an imidazotetrazine-class chemotherapeutic alkylating agent. In the present study, we show that both elevated MPG expression and short hairpin RNA-mediated loss of DNA polymerase beta (Pol beta) expression in human breast cancer cells increases cellular sensitivity to temozolomide. Resistance to temozolomide is restored by complementation of either wild-type human Pol beta or human Pol beta with an inactivating mutation specific to the polymerase active site yet functional for 5'-deoxyribose-phosphate (5'dRP) lyase activity. These genetic and cellular studies uniquely demonstrate that overexpression of MPG causes an imbalance in base excision repair (BER), leading to an accumulation of cytotoxic 5'dRP lesions, and that the 5'dRP lyase activity of Pol beta is required to restore resistance to temozolomide. These results imply that Pol beta-dependent 5'dRP lyase activity is the rate-limiting step in BER in these cells and suggests that BER is a tightly balanced pathway for the repair of alkylated bases such as N7-methylguanine and N3-methyladenine. Furthermore, we find that 5'dRP-mediated cell death is independent of caspase-3 activation and does not induce the formation of autophagosomes, as measured by green fluorescent protein-light chain 3 localization. The experiments presented herein suggest that it will be important to investigate whether an active BER pathway could be partially responsible for the temozolomide-mediated resistance seen in some tumors and that balanced BER protein expression and overall BER capacity may help predict sensitivity to temozolomide.