Recycling of ascorbate from its oxidized forms is essential to maintain stores of the vitamin in human cells. Whereas reduction of dehydroascorbate to ascorbate is thought to be largely GSH-dependent, we reconsidered the possibility that the selenium-dependent thioredoxin system might contribute to ascorbate regeneration. We found that purified rat liver thioredoxin reductase functions as an NADPH-dependent dehydroascorbate reductase, with an apparent Km of 2. 5 mM for dehydroascorbate, and a kcat of 90 min-1. Addition of 2.8 microM purified rat liver thioredoxin lowered the apparent Km to 0.7 mM, without affecting the turnover (kcat of 71 min-1). Since thioredoxin reductase requires selenium, we tested the physiologic importance of this enzyme for dehydroascorbate reduction in livers from control and selenium-deficient rats. Selenium deficiency lowered liver thioredoxin reductase activity by 88%, glutathione peroxidase activity by 99%, and ascorbate content by 33%, but did not affect GSH content. NADPH-dependent dehydroascorbate reductase activity due to thioredoxin reductase, on the basis of inhibition by aurothioglucose, was decreased 88% in dialyzed liver cytosolic fractions from selenium-deficient rats. GSH-dependent dehydroascorbate reductase activity in liver cytosol was variable, but typically 2-3-fold that of NADPH-dependent activity. These results show that the thioredoxin system can reduce dehydroascorbate, and that this function is required for maintenance of liver ascorbate content.