The activation of phospholipase D in different cell lines treated with recombinant human tumor necrosis factor (TNF) has been investigated. When the murine fibrosarcoma cell lines L929 and WEHI164c113, as well as the human promonocytic cell line U937, were prelabeled with [14C]palmitic acid or [3H]arachidonic acid, and treated with TNF in the presence of ethanol, TNF induced the synthesis of [14C]phosphatidylethanol or [3H]phosphatidylethanol, respectively, as the result of a phospholipase-D-catalyzed transphosphatidylation. TNF-induced phospholipase D activity was observed 1 h before the onset of cell killing and gradually increased thereafter. Subclones selected for resistance to TNF cytotoxicity did not show phospholipase D activation in response to TNF. In contrast, when these subclones were treated with TNF in the presence of actinomycin D, TNF cytotoxicity as well as TNF-induced phospholipase D activity could be restored. TNF cytotoxicity and TNF-induced phospholipase D activity were equally modulated by various drugs known to interfere with different steps in the TNF-signaling pathway. Phospholipase D activation was found not to be the result of cell killing per se, as a number of other cytotoxic reagents were unable to activate phospholipase D. Prelabeling of cells with [14C]lysophosphatidylcholine indicated phosphatidylcholine as one of the substrates for TNF-activated phospholipase D. In conclusion, this study demonstrates that TNF-induced cytotoxicity is associated with activation of phospholipase D.