It has been amply documented that L-ascorbic acid added to the medium of a cell culture increases oxidative damage, and this effect of L-ascorbic acid has been ascribed to the generation of reactive oxygen intermediates in the medium during its auto-oxidation. We have here questioned whether such an effect is exerted inside the cell as well, and if so, what its mechanism is. To assess thiol oxidation in the cell, we manipulated CHO cells so that they could express bacterial alkaline phosphatase in the cytoplasm. Alkaline phosphatase activity, which requires the formation of intramolecular disulfide bridges, was shown to appear when the cells were exposed to H₂O₂. This H₂O₂-induced activity increased more than 1.5 fold when L-ascorbic acid had been loaded in the cells by incubation with Lascorbic acid-2-O-phosphate. Similar enhancing effects were also observed by assessing oxidation of glutathione, formation of protein carbonyls, and generation of reactive oxygen intermediates. Interestingly, the effects by the L-ascorbic acid-2-O-phosphate treatment were totally suppressed by addition of the membrane-permeable chelator deferoxamine to the medium, indicating the involvement of iron ions. Because the apoprotein of conalbumin, which binds iron ions with a high affinity, had no effect and because the same deferoxamine effect was observed with the cells incubated in balanced salt solution with no metal salts added, it was concluded that L-ascorbic acid acts as a pro-oxidant within the cell suffering oxidative stress, and that this effect is elicited through increased redox-cycling of iron in combination with L-ascorbic acid.