The cellular events involved in direct tumor-cell destruction by tumor necrosis factor (TNF) are still incompletely understood, but a role of endonucleases has been suggested. In the present study we have analyzed in detail the effect of Zn++, an inhibitor of endonucleases, on TNF-mediated DNA-fragmentation and on cytolysis in Actinomycin-D-pre-treated WEHII 64-S cells. Beginning 2 hr after addition of TNF, a rapid degradation of cellular DNA is observed, as evidenced by release of 3H-Thymidine (TdR) label from nuclei into cytoplasm. TNF-mediated lysis of WEHII64-S cells begins at 3 hr and reaches plateau levels at 7 hr. Addition of Zn++ to TNF-treated WEHII64 cells completely abrogates DNA fragmentation at ImM. Of greater importance is the fact that Zn++ treatment also completely blocks TNF-mediated cytolysis of the target cells. Concentrations-between 0.1 and I mM ZnSO4 prevent cell death, as assessed by chromium-release and Trypan blue dye exclusion. In addition, ZnCl2, but not other divalent cations like CaCl2, MgSO4 and CuSO4 in the same concentration range, prevents cell death as well, demonstrating that the effect in fact is mediated by Zn++. Zn++ added 2 hr after TNF treatment, still effectively inhibits cell lysis, indicating that it acts at a late stage after binding of TNF to its receptor. Our data suggest that activation of endonucleases is not an accompanying effect but an essential step in TNF-mediated tumor-cell destruction.