We reported previously that α-tocopheryl-succinate (VES) induced apoptosis of cultured human promyelocytic leukemia cells (HL-60) (Free Radic Res 2000;33:407-18). We have now studied the effect of cholesteryl-hemisuccinate (CS) on the fate of HL-60 cells to clarify whether CS has an effect similar to that of VES. CS inhibited the growth of HL-60 cells without differentiation to granulocytes and induced DNA fragmentation and ladder formation. CS inhibited the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt) and initiated the activation of a caspase cascade. CS triggered the reaction leading to the cleavage of Bid and also released cytochrome c (Cyt. c) from mitochondria. In addition, CS induced mitochondrial membrane depolarization and translocation of Bax to mitochondria in HL-60 cells. However, CS did not induce an increase in the concentration of intracellular calcium ions in HL-60 cells. The membrane depolarization, Cyt. c release, and DNA fragmentation were inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor, but not by cyclosporin A, an inhibitor of membrane permeability transition. These results suggested that CS-induced apoptosis of HL-60 cells might be caused by inhibiting Akt phosphorylation following cleavage of Bid through caspase-8 activation and subsequently via an Apaf complex-caspase cascade pathway.