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The synthesis of heat shock proteins (HSP) and the development of thermotolerance were studied in Chinese hamster ovary cells in order to determine whether depletion of intracellular glutathione (GSH) inhibited their expression. Cells were exposed to 100 microM diethylmaleate/50 microM buthionine sulfoximine which reduced GSH levels by 95% or more during the experimental time course. HSP synthesis was induced by incubation at 43 degrees C for varying durations. Synthesis was independent of the diethylmaleate/buthionine sulfoximine treatment if mild heat shocks (e.g., 43 degrees C for 15 min) were administered but was suppressed by such severe treatments as 45 or 60 min at 43 degrees C which caused inhibition of non-heat shock protein synthesis. GSH depletion also resulted in inhibition of thermotolerance triggered by a 45-min, 43 degrees C heat shock. This observation and a previous one, which showed that inhibition of protein synthesis by exposure to cycloheximide inhibited both HSP and tolerance (M. L. Freeman et al., Radiat. Res., 112: 564-574, 1987), indicate that glutathione is not involved in either the synthesis of HSP or the expression of tolerance but that GSH depletion can inhibit them indirectly via nonspecific inhibition of protein synthesis.