Ascorbic acid, or vitamin C, generally functions as an antioxidant by directly reacting with reactive oxygen intermediates and has a vital role in defenses against oxidative stress. However, ascorbic acid also has pro-oxidant properties and may cause apoptosis of lymphoid and myeloid cells. The present study shows that dehydroascorbate, the oxidized form of vitamin C, stimulates the antioxidant defenses of cells, preferentially importing dehydroascorbate over ascorbate. While 200-800 microM vitamin C caused apoptosis of Jurkat and H9 human T lymphocytes, pretreatment with 200-1000 microM dehydroascorbate stimulated activity of pentose phosphate pathway enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase, elevated intracellular glutathione levels, and inhibited H(2)O(2)-induced changes in mitochondrial transmembrane potential and cell death. A 3. 3-fold maximal glutathione elevation was observed after 48 h stimulation with 800 microM dehydroascorbate. In itself, dehydroascorbate did not affect cytosolic or mitochondrial reactive oxygen intermediate levels as monitored by flow cytometry using oxidation-sensitive fluorescent probes. The data reveal a novel mechanism for increasing glutathione levels through stimulation of the pentose phosphate pathway and identify dehydroascorbate as an antioxidant for cells susceptible to the pro-oxidant and proapoptotic properties of vitamin C.