Chinese hamster ovary (CHO) cells heated for 10 min at 45 degrees C became thermotolerant to a second heat exposure at either pH 7.3 or 6.6. However, low pH delayed the development of thermotolerance and suppressed the rate of synthesis of all proteins, including heat-shock proteins (HSPs). Low-pH-tolerant mutant CHO cells (PHV2) in pH 6.6 medium were also delayed in both the development of thermotolerance and protein synthesis, though less than CHO cells in pH 6.6 medium. The rate of synthesis of inducible HSP-70 under the three conditions paralleled the kinetics of the development of thermotolerance. The intracellular pH (pHi) of CHO cells in pH 6.6 medium, whether heated or not, was far lower than CHO cells in pH 7.3 medium, and the pHi of PHV2 cells in pH 6.6 medium was close to that of CHO cells in pH 7.3 medium after the initial heat shock. Amiloride enhanced the effect of low-pH medium on pHi HSP synthesis and development of thermotolerance. The concentration of HSP-70 was also measured by flow cytometry. The level of HSP-70 was not altered within the first 4 h after a 10-min 45.0 degrees C initial heat treatment during which the major portion of thermotolerance developed, though the level of HSP-70 increased rapidly after 4 h. Low pH caused a further delay in the increase in HSP-70. We conclude that low-pH medium may inhibit the synthesis of HSPs in part by lowering pHi, but the delay and suppression of development of thermotolerance is not primarily due to the inhibition of HSP synthesis.