The induction of cell death in leukemic HL‐60 cells by the ether lipid 1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycero‐3‐phosphocholine (ET‐18‐OCH3; 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‐OCH3 induced disruption of the mitochondrial transmembrane potential (ΔΨm) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET‐18‐OCH3 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‐OCH3 analogues unable to induce apoptosis failed to disrupt ΔΨm and to activate caspase‐3. ET‐18‐OCH3‐resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase‐3 activation and did not undergo ΔΨm disruption upon ET‐18‐OCH3 incubation. Cyclosporin A partially inhibited ΔΨm dissipation, caspase activation and apoptosis in ET‐18‐OCH3‐treated leukemic cells. Overexpression of bcl‐2 by gene transfer prevented ΔΨm collapse, ROS generation, caspase activation and apoptosis in ET‐18‐OCH3‐treated leukemic T cells. Pretreatment with the caspase inhibitor Z‐Asp‐2,6‐dichlorobenzoyloxymethylketone prevented ET‐18‐OCH3‐induced PARP proteolysis and DNA fragmentation, but not ΔΨm dissipation. ET‐18‐OCH3 did not affect the expression of caspases and bcl‐2‐related genes. ET‐18‐OCH3‐induced apoptosis did not require protein synthesis. Our data indicate that ΔΨm dissipation and caspase‐3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET‐18‐OCH3, and that the sequence of events in the apoptotic action of ET‐18‐OCH3 on human leukemic cells is: ΔΨm disruption, caspase‐3 activation and internucleosomal DNA degradation. Int. J. Cancer 86:208–218, 2000. © 2000 Wiley‐Liss, Inc.