Pancreatic beta cells are susceptible to oxidative stress, which is related closely to the islet dysfunction. In the present study, using the pancreatic cell lines HIT-T15 and RINm5F as known in vitro models of impaired beta cell function as well as primary rat islet beta cells, we observed a relationship between intracellular glutathione levels and oxidative stress-mediated cell dysfunction. Hydrogen peroxide and 4-hydroxy-2-nonenal caused cell death in HIT-T15 and RINm5F cells at lower concentrations compared to non-beta cells, such as HepG2 and NRK-49F cells. The extent of the cytotoxicity caused by the model oxidants was inversely correlated well with intracellular glutathione levels in the cell lines used. Treatment of HIT-T15 and RINm5F cells with L-cysteine or L-cystine significantly augmented the glutathione contents, surpassing the effect of N-acetylcysteine, and abrogated 4-hydroxy-2-nonenal -mediated cytotoxicity almost completely. L-Cysteine increased intracellular glutathione levels in primary beta cells as well. Supplementation of L-cysteine to the RINm5F cell culture inhibited 4-hydroxy-2-nonenal-mediated cytosolic translocation of PDX-1, a key transcription factor for beta cell function. Intrinsic transport activities (Vmax/Km) of the L-cystine/L-glutamate exchanger in HIT-T15 and RINm5F cells were considerably lower than that in NRK-49F cells although gene expressions of the exchanger were similar in these cells. Results obtained from the present study suggest that the restricted activity of the L-cystine/L-glutamate exchanger controls the levels of intracellular glutathione, thereby making beta cells become susceptible to oxidative stress.