The antitumor ether lipid ET-18-OCH₃ promotes apoptosis in tumor cells through intracellular activation of Fas/CD95. Results of this study showed that ET-18-OCH₃ induces cocapping of Fas and membrane rafts, specialized plasma membrane regions involved in signaling, before the onset of apoptosis in human leukemic cells. Patches of membrane rafts accumulated Fas clusters in leukemic cells treated with ET-18-OCH₃. Sucrose gradient centrifugation of Triton X-100 cell lysates showed that Fas translocated into membrane rafts following ET-18-OCH₃ treatment of T-leukemic Jurkat cells. Disruption of membrane raft integrity by methyl-β-cyclodextrin or filipin inhibited ET-18-OCH₃-induced apoptosis in leukemic primary cells and cell lines. Fas clustering was also inhibited by methyl-β-cyclodextrin. These data indicate that ET-18-OCH₃ reorganizes membrane rafts to trigger apoptosis in human leukemic cells, and that Fas coaggregation with membrane rafts is required for ET-18-OCH₃–induced apoptosis. This translocation of Fas into membrane rafts may provide a mechanism for amplifying Fas signaling by reorganization of membrane microdomains.