The cytokine tumor necrosis factor (TNF) activates diverse signaling molecules resulting in gene expression, differentiation, and/or cell death. Here we report a novel feature induced by TNF, namely translocation of mitochondria from a dispersed distribution to a perinuclear cluster. Mitochondrial translocation correlated with sensitivity to the cell death-inducing activity of TNF and was mediated by the 55-kDa TNF receptor (TNF-R55), but not by Fas, indicating that the signaling pathway requires a TNF-R55-specific but death domain-independent signal. Indeed, using L929 cells that express mutant TNF-R55, we showed that the membrane-proximal region of TNF-R55 was essential for signaling to mitochondrial translocation. In the absence of translocation, the cell death response was markedly delayed, pointing to a cooperative effect on cell death. Translocation of mitochondria, although dependent on the microtubules, was not imposed by the latter and was equally induced by TNF-independent immunoinhibition of the motor protein kinesin. Additionally, immunoinhibition with antibody directed against the tail domain of kinesin synergized with TNF-induced cell death. Based on this functional mimicry, we propose that a TNF-R55 membrane-proximal region-dependent signal impedes mitochondria-associated kinesin, resulting in cooperation with the TNF-R55 death domain-induced cytotoxic response and causing the observed clustering of mitochondria.