Using agonistic antibodies (Ab) we have examined whether the 75-kDa chain of the tumor necrosis factor receptor (p75 TNFR) is capable of mediating cytotoxic response and gene regulation alone or in cooperation with p55 TNFR. Addition of an anti-p75 TNFR polyclonal antiserum or anti-p75 monoclonal antibody (mAb) plus anti-immunoglobulin (Ig) led to cytotoxic response of human KYM-1 rhabdomyosarcoma cells. Anti-p75 mAb alone had no effect pointing out the importance of strong receptor stimulation for signal transduction into the cell. Simultaneous triggering of both the p55 and p75 TNFR by agonistic Ab resulted in additive cytotoxic action on KYM-1 cells. The anti-p75 mAb 3H5, directed to a non-TNF binding site on the human p75 TNFR, was used to confirm further the ability of the p75 TNFR to potentiate p55 TNFR-mediated cell death. While exhibiting no cytotoxicity by its own, 3H5 significantly augmented the cytotoxic effect of the anti-p55 mAb htr9 towards KYM-1 cells. Neither the anti-p75 TNFR antiserum nor anti-p75 mAb were cytotoxic for human U937 cells suggesting that the cytolysis resulting from p75 TNFR cross-linking is cell specific. Noteworthy, stimulation of the p75 TNFR with mAb plus anti-Ig or polyclonal antiserum led to a marked enhancement of the p55 TNFR-induced U937 cell death, indicating collaboration between the two TNFR in induction of cytotoxicity also in this cell line. However, 3H5 mAb did not affect the ability of anti-p55 mAb to lyse U937 cells. Altogether, these data demonstrate the difference between KYM-1 and U937 cell lines with respect to the role for the p75 TNFR in mediating cytotoxicity. Both TNFR were found to mediate cytomegalovirus (CMV) promoter activation in human SW480T-beta Gal cells and nuclear transcription factor kappa B (NF-kappa B) induction in this cell line as well as in KYM-1 cells. It was demonstrated for the first time that independent stimulation of both TNFR resulted in an additive effect on the CMV promoter activation and induction of the NF-kappa B. Taken together, these results indicate that the p75 TNFR induces cytotoxicity in a cell-specific manner and potentiates p55 TNFR-mediated cytotoxic response and gene regulation.