After cells have been exposed to a nonlethal heat shock, they develop an enhanced resistance to subsequent prolonged heat shock. This process, termed thermotolerance, correlates with the expression of a group of proteins called the heat shock proteins. When cells are exposed to heat, protein synthesis is rapidly turned off and takes 5-6 hr to recover. In thermotolerant cells, protein synthesis is not blocked by heat. The heat shock proteins are thought to be responsible for the development of therrnotolerance and the protection of the protein synthesis machinery from heat inactivation. To test the hypothesis that the heat shock proteins are involved in the heat shock response, we used two inhibitors to block their transcription and expression during heating and then monitored the effect on the development of thermotolerance and on protein synthesis. Carnptothecin inhibits DNA topoisomerase I and blocks transcription of all actively transcribed genes, whereas dichloro-D-ribofuranosylbenzimidazole (DRB) inhibits only those genes transcribed by RNA polymerase II. Both DRB and carnptothecin blocked the heat-induced expression of the heat shock proteins, but the absence of these proteins did not block either the development of thermotolerance or the protection of protein synthesis after heating. The data indicate that thermotolerance can develop in the absence of new protein synthesis. Q 1992 ~i ~e y -~i s s , Inr.