We compared the biological function of the human tumor necrosis factor receptors p55 (hTNF-R55) and p75 (hTNF-R75) expressed in the murine (m) fibrosarcoma cell line L929. Receptor-specific triggering of hTNF-R55 in transfected L929 cells by agonistic monoclonal antibodies or hTNF-R32WS86T, a hTNF-R55-specific mutant of hTNF, resulted in cytotoxicity. Specific clustering of hTNF-R75 in transfected L929 cells by agonistic monoclonal antibodies or hTNF-D143F, a hTNF-R75-specific mutant of hTNF also induced cytotoxicity, albeit at low level. In both cases, the cytotoxic activity of receptor clustering could be synergized by addition of 20 mM LiCl. Remarkably, cytotoxicity induced after R75 triggering in transfected L929 cells could be completely abolished by addition of neutralizing anti-mTNF antibodies, in contrast to cell killing seen after specific R55 clustering. No soluble mTNF could be demonstrated using a sensitive biological assay, although L929 cells were expressing low levels of mTNF-specific mRNA as shown by PCR. These data clearly demonstrate that minute amounts of endogenously produced TNF can be a key mediator in R75-mediated cytotoxicity. Presumably, the latter efficiently traps the ligand and transfers it to TNF-R55, and/or by binding it, protects the endogenously made TNF from inactivation.