Glutathione (GSH) and ascorbate, the cytoplasmic antioxidants, can regenerate and replace each other in scavenging reactive oxygen species reaction. Mice, but not guinea pigs, produce ascorbate endogenously. L-Buthionine-S,R-sulfoximine (L-S,R-BSO) substantially inhibited GSH production at a greater degree and caused a higher toxicity to guinea pigs than mice, implying that mice may have an additional protective mechanism against oxidative stress injury. Indeed, administration of L-S,R-BSO to mice inhibited tissue GSH production while increasing ascorbate levels. L-S,R-BSO also increased tissue ascorbate levels in mice fed a ascorbate and dehydroascorbate-free diet suggesting activation of ascorbate synthesis, which was further confirmed by increased urinary ascorbate excretion. Other reagents inhibiting GSH production also increased tissue ascorbate levels. The results of Northern blot and promoter assay showed that L-S,R-BSO increased mRNA expression and promoter activity of mouse liver L-gulono-gamma-lactone oxidase, the critical enzyme for ascorbate synthesis. Taken together, our study demonstrates that inhibition of GSH production activates ascorbate synthesis to protect mice against oxidative stress injury, the mechanism which is not present in guinea pigs or humans. (C) 2017 Elsevier Inc. All rights reserved.