Culturing Chinese hamster ovary cells in low pH (6.6) medium for several months altered the reproductive survival of these cells to combined low pH treatments and 42.0 degrees C heating. We isolated new pH-resistant cells (identified as pHV-2) with enhanced ability to grow and divide under a low pH (6.6) environment. Their growth characteristics include (a) a plating efficiency of 70%, (b) a doubling time of 16 to 17 h, and (c) a steady state intracellular pH 0.12 pH units higher than for cells grown at a normal pH of 7.3. The pHV-2 cells had 100- to 200-fold increases in survival after 5 h of heating compared to cells incubated at low pH (6.6) for 4 h prior to and during the heat treatments. In addition, they developed a significant degree of thermotolerance. We measured a progressive decline in the intracellular pH as a function of time at 42.0 degrees C. However, the decrease in the intracellular pH did not seem to be correlated with the increased heat sensitivity. The ability to select for low pH variants may have important implications in the extrapolation of in vitro hyperthermic data to the in vivo situation.