A minority of transformed cell lines are directly susceptible to lysis by TNF, whereas many cells can be made sensitive to TNF by treatment with inhibitors of protein synthesis. Other groups have shown that exposure to TNF induces in many cells a transcription/translation dependent response that protects the cell from TNF lysis. Heat shock proteins are involved in protecting cells from the lethal affects of heat and other metabolic poisons. In this report, we test the possibility that heat shock proteins are also involved in protecting cells from lysis by TNF. We find that after induction of the cellular heat shock response by either heat or arsenite treatment, both spontaneously TNF-sensitive cells and those cells made sensitive by inhibition of protein synthesis are nearly completely protected from TNF cytolysis. The heat-treated cells retained most of their capacity to bind TNF, suggesting that heat shock functions at a postreceptor binding phase of the lytic process. Mouse C3HA fibroblasts are also made sensitive to TNF lysis by treatment with cytochalasin E. We have previously found that elicitation of the cell's TNF-protective response by exposure to TNF suppresses killing of C3HA by subsequent treatment with TNF plus cytochalasin E. In contrast, we report here that induction of the heat shock response did not provide significant protection to C3HA from killing by TNF in the presence of cytochalasin E. Thus, although induction of heat shock proteins does protect cells from TNF, they appear to act by a mechanism distinct from that elicited by TNF itself.