We have recently shown that major heat shock protein (hsp70) protects WEHI-S tumor cells from the cytotoxicity mediated by TNF. In the present study, the mechanism of hsp70-associated TNF resistance was investigated. Overexpression of human hsp70 or inhibition of endogenous hsp70 synthesis by expression of antisense hsp70 RNA did not change the ability of WEHI-S tumor cells to bind TNF or internalize and degrade the receptor-bound TNF. Moreover, TNF-induced activation of NF-kappa B-like transcription factors was unaffected by altered levels of hsp70 as tested by electrophoretic mobility shift assay. Thus, it is unlikely that the resistance is due to changes in TNF receptors or in their ability to transduce signals leading to the regulation of genes, whose expression is regulated by NF-kappa B-like transcription factors. The idea that hsp70-associated TNF resistance is independent of regulation of TNF-induced gene expression was further supported by the results showing that hsp70 protected WEHI-S cells from TNF-mediated killing also in the presence of inhibitors of either translation or transcription. Interestingly, TNF-induced activation of arachidonic acid metabolism correlated directly with their sensitivity to TNF and inversely with the amount of hsp70 in the cells. Furthermore, TNF-induced activation of arachidonic acid metabolism was inhibited in WEHI-S cells and two TNF-sensitive human cell lines by induction of the synthesis of endogenous heat shock proteins by heat shock. Even stronger inhibition of arachidonic acid metabolism was seen in WEHI cells rendered TNF-resistant by culturing them in the presence of increasing concentrations of TNF. These cells also had reduced numbers of type 1 TNF receptors. Overexpression of a low molecular weight heat shock protein hsp27 in WEHI-S cells had no effect on any of the parameters studied. These results show that both hsp70-mediated and TNF-induced TNF resistance are associated with a reduced activation of phospholipase A2 suggesting that phospholipase A2 plays an essential role in TNF-mediated cytotoxicity and that hsp70 interferes with the signal transduction pathway leading to its activation.