Chinese hamster ovary cells cultured in vitro were exposed to hyperthermic treatment at 43 degrees C or 45 degrees C. Depletion of 99.5% of the intracellular glutathione (GSH) by addition of 0.1 mM diethylmaleate (DEM) increased thermal sensitivity. This effect, however, was dependent upon oxygen tension. Decreasing the oxygen tension reduced the effect. Thermal cytotoxicity was further augmented by continued depletion of GSH after hyperthermic treatment. Once cells became thermally tolerant, depletion of GSH did not affect thermal sensitivity. The development of thermotolerance was triggered by exposing Chinese hamster ovary cells to a thermal dose consisting of either 30 min at 43 degrees or 10 min at 45 degrees. After a 4-h incubation at 37 degrees, the cells were reheated at 43 degrees or 45 degrees, respectively. A triggering dose of 30 min at 43 degrees is a nonlethal treatment, while 10 min at 45 degrees kills 50% of the cells. Cells were exposed to 0.1 mM DEM during the triggering and development phases of tolerance. Depletion of GSH by addition of DEM did not inhibit the development of thermal tolerance when triggered by the 30-min-43 degrees thermal dose. However, exposure to DEM did inhibit the development of thermal tolerance if the triggering dose consisted of 10 min at 45 degrees. These results were interpreted to mean that GSH depletion will inhibit the development of thermotolerance only after a sufficiently toxic thermal dose.