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Vesnarinone is an effective inotropic agent for treating congestive heart failure, but its clinical usage is restricted because of the severe side effect of agranulocytosis. In myeloid HL-60 cells, vesnarinone increased the intracellular content of a proapoptotic lipid mediator, ceramide, in a time- and dose-dependent manner. Vesnarinone-induced apoptosis was significantly enhanced by simultaneous treatment with a cell-permeable N-acetyl sphingosine (C2-ceramide). Treatment with neither vesnarinone, C2-ceramide, nor simultaneously with vesnarinone and C2-ceramide caused a marked increase of reactive oxygen intermediates (ROI) generation measured by the 2',7'-dichlorofluorescin method. However, oxidative damage judged by the production of lipid peroxidates and the nitroblue tetrazolium-reducing ability were enhanced more significantly by simultaneous treatment with vesnarinone and C2-ceramide than by vesnarinone alone. Moreover, vesnarinone inhibited catalase function both at the protein and activity level, and this inhibition was synergistically enhanced by C2-ceramide, and vesnarinone-induced oxidative damage and apoptosis were significantly suppressed by treatment of HL-60 cells with purified catalase. C2-ceramide enhanced vesnarinone-induced inhibition of the ROI-scavenging enzyme catalase at the levels of protein and activity in HL-60 cells; in contrast, however, vesnarinone did not induce ceramide generation, oxidative damage, or catalase depletion in HL-60/ves cells, where vesnarinone could not induce apoptosis. Taken together, the results suggest that vesnarinone induces myeloid cell apoptosis by increasing oxidative damage via ceramide-induced inhibition of catalase function.