Increasing evidence suggests that cancer cells (relative to normal cells) have altered mitochondrial electron transport chains (ETC) that are more likely to form reactive oxygen species (ROS; i.e., O(2)(*-) and H(2)O(2)) resulting in a condition of chronic metabolic oxidative stress, that maybe compensated for by increasing glucose and hydroperoxide metabolism. In the current study, the ability of an inhibitor of glucose metabolism, 2-deoxy-D-glucose (2DG), combined with mitochondrial electron transport chain blockers (ETCBs) to enhance oxidative stress and cytotoxicity was determined in human colon cancer cells. Treatment of HT29 and HCT116 cancer cells with Antimycin A (Ant A) or rotenone (Rot) increased carboxy-dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidine (DHE) oxidation, caused the accumulation of glutathione disulfide and enhanced 2DG-induced cell killing. In contrast, Rot did not enhance the toxicity of 2DG in normal human fibroblasts supporting the hypotheses that cancer cells are more susceptible to inhibition of glucose metabolism in the presence of ETCBs. In addition, 2-methoxy-antimycin A (Meth A; an analog of Ant A that does not have ETCB activity) did not enhance 2DG-induced DHE oxidation or cytotoxicity in cancer cells. Finally, in HT29 tumor bearing mice treated with the combination of 2DG (500 mg/kg) + Rot (2 mg/kg) the average rate of tumor growth was significantly slower when compared to control or either drug alone. These results show that 2DG-induced cytotoxicity and oxidative stress can be significantly enhanced by ETCBs in human colon cancer cells both in vitro and in vivo.