The characteristic development of thermotolerance was examined in tsAF8 cells, which were temperature sensitive cell line derived from BHK21. tsAF8 cells lose RNA polymerase II activity and arrest in the G(1) phase of the cell cycle at the nonpermissive temperature of 39.5-40.6 degrees C. tsAF8 cells were pre-heated at 45 degrees C f or 20 min, incubated at 34 or 39.7 degrees C for different durations, and then heated again at 45 degrees C for 30 min. The cells were trypsinized immediately after the second heating to evaluate cell survival. As a result, thermotolerance was developed rapidly in the cells incubated at 34 degrees C. However, the survival rate of the cells incubated at 39.7 degrees C was almost constant. Thermotolerance of BHK21 cells, which were wild-type cells of tsAF8 cells, developed at 39.7 degrees C. These results suggest that thermotolerance may be inhibited in tsAF8 cells incubated at 39.7 degrees C, and that some metabolism which is inhibited in tsAF8 cells at 39.7 degrees C may be related to thermotolerance development.