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Pharmacological ascorbate, via its oxidation, has been proposed as a pro-drug for the delivery of H2O2 to tumors. Pharmacological ascorbate decreases clonogenic survival of pancreatic cancer cells, which can be reversed by treatment with scavengers of H2O2. The goal of this study was to determine if inhibitors of intracellular hydroperoxide detoxification could enhance the cytotoxic effects of ascorbate. Human pancreatic cancer cells were treated with ascorbate alone or in combination with inhibitors of hydroperoxide removal including the glutathione disulfide reductase inhibitor 1,3 bis (2-chloroethyl)-1-nitrosurea (BCNU), siRNA targeted to glutathione disulfide reductase (siGR), and 2-deoxy-D-glucose (2DG), which inhibits glucose metabolism. Changes in the intracellular concentration of H2O2 were determined by analysis of the rate of aminotriazole-mediated inactivation of endogenous catalase activity. Pharmacological ascorbate increased intracellular H2O2 and depleted intracellular glutathione. When inhibitors of H2O2 metabolism were combined with pharmacological ascorbate the increase in intracellular H2O2 was amplified and cytotoxicity was enhanced. We conclude that inclusion of agents that inhibit cellular peroxide removal produced by pharmacological ascorbate leads to changes in the intracellular redox state resulting in enhanced cytotoxicity. [ABSTRACT FROM AUTHOR]