Hyperthermia sensitizes tumor cells to killing by tumor necrosis factor-alpha (TNF). Sensitization is greater in cells exposed to TNF before heating begins than with the reverse sequence, and heat-shock proteins (hsp) have been suggested to protect cells from TNF cytotoxicity. Here we examined the role of Hsp27 in TNF resistance. Murine L929 cells were stably transfected with the vector pRc/CMV constitutively to express an inserted human hsp27 complementary DNA (cDNA) sequence. Parental cells produced no detectable murine homolog to human hsp27. Hsp27-sense clones expressed hsp27 messenger RNA (mRNA) and protein at 37 degrees C. Cells transfected with the cDNA in the anti-sense orientation produced anti-sense mRNA but no protein, and cells transfected with the vector alone produced neither product. Expression of hsp27 conferred significant resistance to TNF cytotoxicity in both neutral red cytotoxicity and clonogenic survival assays. Vector along and hsp27 anti-sense transfectants had a TNF response similar to that of parental L929 cells. Kinetic studies in L929 cells showed that hsp27-expressing clones exhibited resistance relative to parental cells beginning 6 h after TNF exposure, and this differential response increased by 12 and 24 h. Addition of actinomycin D to the TNF cytotoxicity assays accelerated the cytotoxicity development in parental and transfected cells, but the hsp27-sense clones were still more resistant. Hsp27-sense clones of L929 cells were also resistant to oxidative stress induced by menadione and released less arachidonic acid in response to TNF induction. These results show that hsp27 can negatively regulate the TNF cytotoxic mechanism.