Chinese hamster V79 cells were made thermotolerant by either continuous heating at 42.5° or by fractionated 43° exposures with interfraction incubation at 37°. For both methods of thermotolerance induction, elevations in cellular glutathione (GSH) were observed. Additionally, GSH was also shown to be elevated following a 1-hr exposure to 6% ethanol, which also induces thermotolerance. These elevations in cellular GSH preceded thermotolerance induction in regard to cell survival. To determine if a reduction in cellular GSH prior to or during heating at 42.5° would influence thermotolerance, GSH levels were reduced by either pretreatment with diethylmaleate, an agent that binds GSH, or treatment during heating with buthionine sulfoximine, an agent that inhibits GSH synthesis. Both depleting protocols resulted in thermosensitization. These data suggest that GSH may be important in the early cellular response to thermal stress.