Chinese hamster ovary (CHO) cells were heated at either pH 7.2 to 7.4 or 6.7 to 6.8 in order to determine if conditions which suppress the development of thermotolerance (pH 6.7 to 6.8) reduce intracellular levels of glutathione (GSH). When the pH of the growth medium was reduced from 7.2 to 6.7, a 25 to 30% reduction in GSH was observed in cells maintained at 37 degrees. Cells heated at 42 degrees in medium adjusted to pH 6.7 had lower levels of GSH compared to cells heated at pH 7.2. Cells were also heated for 1 hr at 43 degrees and then incubated at 37 degrees for up to 9.5 hr prior to GSH measurement. The GSH levels of cells treated at pH 7.3 increased approximately 20% above control, whereas treatment at pH 6.7 resulted in a 20% reduction compared to control. Chinese hamster ovary cells were exposed to 5 mM buthionine sulfoximine (BSO) prior to and during 42 degrees heat treatment. BSO exposure at either pH 7.3 or 6.8 reduced the GSH concentration to approximately 65% of control and increased thermal cytotoxicity. The thermal sensitivity of cells incubated at 42 degrees and pH 7.3 was compared to that of cells incubated at pH 6.8. Decreasing the pH from 7.3 to 6.8 increased sensitivity by a factor of 1.87 in the absence of BSO, whereas decreasing the pH in the presence of BSO increased sensitivity by only 1.50. In summary, these results suggest that the increase in thermal sensitivity observed when Chinese hamster ovary cells are heated in acid medium is due partly to the depletion of GSH.