Abstract There remains great interest in strategies to increase the efficacy of radiation therapy in cancer patients. Glucose depletion occurs commonly in certain regions of bulky solid tumors, owing to high glucose metabolism combined with poor perfusion caused by a disorganized blood vasculature. Evidence suggests that cancer cells located in glucose-deprived regions are resistant to therapeutic killing. Agents that sensitize glucose-deprived cancer cells might offer useful adjuvants for tumor radiotherapy. In an earlier study, we demonstrated that rodent cells which are genetically deficient in the oxidative pentose phosphate cycle (OPPC) display a heightened response to hydroxyethyldisulfide (HEDS), a thiol-specific oxidant that can radiosensitize cells by blocking the redox-dependent function of DNA repair protein Ku. Since glucose is the chief substrate of the OPPC, and the OPPC is responsible for detoxifying HEDS, we reasoned that glucose deprivation should phenocopy the effects of OPPC deficiency in enhancing radiosensitization by HEDS. To test this prediction, we compared the effects of HEDS treatment in human colon cancer cell lines HCT116 or HT29, which are radiation sensitive or resistant, respectively. In both cell lines, HEDS was detoxified by OPPC-mediated conversion to mercaptoethanol (ME) in high-glucose media but not in low-glucose media. Consistent with the decrease in cellular bioreductant activity by glucose depletion, HEDS greatly decreased free glutathione levels under low-glucose conditions. Similarly, HEDS greatly decreased the free thiol content of total proteins and impaired the Ku DNA binding activity in low-glucose conditions. Most significantly, in clonogenic cell growth assay, the cytotoxic effects of HEDS were greatly increased under low glucose conditions, when added by itself to HCT116 cells but particularly when added in combination with irradiation to HCT116 or HT29 cells. Together, our findings indicate that glucose depletion enhances the ability of HEDS to radiosensitize cancer cells. More broadly, our findings suggest the potential to use HEDS in a novel strategy to enhance killing of radioresistant regions of tumors that are characterized by a glucose-depleted state. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1392.