Incubation of Chinese hamster ovary cells in pH 6.6 medium for 4 h prior to and during 42.0 degrees C heating enhanced thermal cell killing compared to cells heated under normal pH 7.3 conditions. We examined the relationship between the extracellular pH and intracellular pH (pHi) of Chinese hamster ovary cells using a flow cytometer with the pH-sensitive fluorescent molecule 2,3-dicyanohydroquinone. Using either normal (7.3) or low (6.6) pH conditions, the mean pHi and population pHi heterogeneity was studied as a function of time at 42.0 degrees C. Cells incubated at pH 6.6 for 4 h had a resting pHi 0.14 to 0.19 pH units lower than cells at normal pH 7.3, indicating the presence of an active pHi regulatory system. Heating 1 h at 42.0 degrees C at normal pH caused an increase in the pHi of 0.14 pH units. With further heating the cells gradually returned to the unheated (7.3) control levels. Similar pHi changes were observed with the cells incubated and heated at pH 6.6. However, the mean pHi was always more acidic than cells heated at normal pH. Active pHi regulation was still possible for a substantial (greater than 30%) number of cells even after 10 h of heating under low pH conditions. These results suggest that a breakdown in pHi regulation is not the mechanism of low pH-induced heat sensitization.