This investigation tested the hypothesis that depletion of intracellular glutathione, in contrast to its oxidation could lead to non-native oxidation of protein thiols, thereby trapping proteins in an unstable conformation. Chinese hamster cells were exposed to the alpha, beta-unsaturated dicarboxylic acid diethylmaleate in order to produce rapid glutathione (GSH) depletion without oxidation. Measurement of the fluorescence of oxidized 2',7'-dichlorofluorescein diacetate indicated that reactive oxygen species accumulated in GSH depleted cells. Glutathione depletion was found to alter protein thiol/disulfide exchange ratios such that 17 to 23 nmol of protein SH/mg protein underwent oxidation. Differential scanning calorimetry (DSC) of glutathione depleted cells yielded a profile of specific heat capacity versus temperature that was characteristic of cells containing destabilized and denatured protein. In addition, cells depleted of glutathione exhibited a two-fold increase in NP-40 insoluble protein. These results indicate that depletion of intracellular glutathione caused oxidation of protein thiols, protein denaturation and aggregation and provide a mechanism to explain how GSH depletion can initiate stress responses.