Sphingolipid metabolites in HL-60 cells were analyzed to gain an understanding of their roles in early events underlying hydrogen peroxide (H2O2)-induced apoptosis. Incubation of cells with H2O2 increased the intracellular levels of ceramides and sphinganine, but decreased those of ceramide 1-phosphates (ceramide 1-P) and sphingosine. The levels of sphingomyelins and sphingomyelinase (SMase) activities were not affected by H2O2 treatment. These results were similar to the profiles induced by daunorubicin, an activator of serine palmitoyl CoA transferase (SPT), suggesting that H2O2 stimulated the de novo synthetic pathway of ceramides. L-cycloserine and fumonisin B1 (FB1), specific inhibitors of de novo ceramide biosynthesis, suppressed the elevation of ceramides and sphinganine induced by H2O2, which consequently reduced apoptotic cell death. Collectively, these results demonstrated that H2O2 increased the intracellular concentrations of ceramides via activation of a de novo biosynthetic pathway, and the enhanced ceramides might initiate apoptosis in HL-60 cells.