Intracellular glutathione (GSH) concentrations were titrated in Chinese hamster ovary cells by exposure to various concentrations of diethylmaleate (DEM). The various steady state levels of GSH obtained were maintained throughout the experimental time course. Cells were incubated at 42 degrees after DEM addition in order to produce thermal dose response curves using colony formation as the end point. The slope of the dose response curve was subsequently determined and compared to the intracellular GSH concentration. This comparison indicated Chinese hamster ovary cells contain multiple reservoirs of GSH which in turn regulate thermal toxicity in a stepwise manner. Removal of 50% or less of the GSH did not affect thermal sensitivity. A small increase in sensitivity occurred when 50 to 80% of the GSH was removed. Removal of greater than 80% of the GSH increased thermal toxicity significantly. The facts that 10 and 20 microM DEM produce extensive GSH depletion and only small changes in survival imply that a threshold concentration of GSH must be removed before thermal toxicity is affected.