The induction of cell death in leukemic HL-60 cells by the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3phosphocholine (ET-18-OCH 3 ; edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET-18-OCH 3 induced disruption of the mitochondrial transmembrane potential (⌬⌿ m ) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET-18-OCH 3 also induced caspase-3 activation in human leukemic cells, as assessed by cleavage of caspase-3 into the p17 active form and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). ET-18-OCH 3 analogues unable to induce apoptosis failed to disrupt ⌬⌿ m and to activate caspase-3. ET-18-OCH 3 -resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase-3 activation and did not undergo ⌬⌿ m disruption upon ET-18-OCH 3 incubation. Cyclosporin A partially inhibited ⌬⌿ m dissipation, caspase activation and apoptosis in ET-18-OCH 3 -treated leukemic cells. Overexpression of bcl-2 by gene transfer prevented ⌬⌿ m collapse, ROS generation, caspase activation and apoptosis in ET-18-OCH 3 -treated leukemic T cells. Pretreatment with the caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone prevented ET-18-OCH 3 -induced PARP proteolysis and DNA fragmentation, but not ⌬⌿ m dissipation. ET-18-OCH 3 did not affect the expression of caspases and bcl-2-related genes. ET-18-OCH 3 -induced apoptosis did not require protein synthesis. Our data indicate that ⌬⌿ m dissipation and caspase-3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET-18-OCH 3 , and that the sequence of events in the apoptotic action of ET-18-OCH 3 on human leukemic cells is: ⌬⌿ m disruption, caspase-3 activation and internucleosomal DNA degradation.