Chinese hamster ovary cells were exposed to various concentrations of diethylmaleate in order to produce various levels of intracellular glutathione (GSH) depletion. Exposure to a 20 microM concentration or more of diethylmaleate depleted the intracellular glutathione concentration by 80% or more and resulted in enhanced synthesis of two 32 kDa proteins which exhibited a pI of about 6.5. Exposure of cells to 50 microM buthionine sulfoximine for 24 hr reduced GSH levels by 95% but did not enhance the synthesis of this protein. Addition of diethylmaleate to buthionine sulfoximine-treated cells resulted in enhanced synthesis of the 32 kDa protein however. Exposure to 0.4 mM diamide triggered the synthesis of several heat shock proteins but did not induce the synthesis of the 32 kDa protein. These results indicated that enhanced synthesis of the 32 kDa protein occurred only after glutathione depletion exceeded 80% and required formation of a glutathione conjugate.