The human rhabdomyosarcoma cell line KYM-1 coexpresses high numbers of both tumor necrosis factor-alpha (TNF) receptors, TR60 and TR80, and is highly sensitive to TNF-induced cytotoxicity. We show here that each receptor type can mediate cytotoxicity on its own on selective stimulation. This was achieved using receptor specific antibodies, able to compete with ligand binding to the respective receptor molecules. Thus, the TR60-specific monoclonal antibody H398 was strongly agonistic, i.e., cytotoxic, in the presence of a secondary cross-linking immunoglobulin. Selective stimulation of TR80 by antibody-mediated receptor crosslinking also induced strong cytotoxicity in KYM-1 cells. Interestingly, when both receptor subsets were stimulated in parallel by limited receptor cross-linking, additive cytolytic effects were observed. In each case the respective Fab fragments showed no agonistic activity. When the natural ligand TNF was used to induce cytolysis, blocking studies with receptor specific Fab fragments indicated that the main cytotoxic effect of TNF is mediated via TR60, although these receptors represent only about 8% of the total TNF receptor number.