Dehydroascorbate (DHA) is a higher oxidation state of ascorbate formed through its action as an intracellular antioxidant. The recycling of DHA back to ascorbate is thought to be catalyzed by a variety of enzymes, including protein disulfide isomerase (PDI), linking ascorbate metabolism to oxidative protein folding in the endoplasmic reticulum (ER). Here we examine the possible role of PDI as a dehydroascorbate reductase. We find the reaction too slow to be the major route for reduction of DHA in the ER, with a second-order rate constant for the reaction of reduced PDI with DHA of only 12.5 M(-1)s(-1). Rates of a similar order of magnitude were obtained for other thioredoxin-superfamily members. However, glutaredoxin was able to catalyze DHA reduction more rapidly through a monothiol mechanism. In addition, DHA can rapidly react with many other dithiol systems, including dithiols in unfolded or partially folded proteins in a PDI-independent manner, with second-order rate constants of up to 186 M(-1)s(-1). Furthermore, we identify borate as a potent inhibitor of catalyzed and noncatalyzed DHA reduction in vitro. This study both provides insights into the link between ascorbate metabolism and oxidative protein folding and suggests a novel link between ascorbate metabolism and borate toxicity.