The present study is aimed to determine the role of Ca2+ signaling evoked by hydrogen peroxide (H2O2) on caspase activation in human leukemia cell line HL-60. We have analysed cytosolic free Ca2+ concentration ([Ca2+]c) determination, mitochondrial membrane potential and caspase-3 and -9 activity by fluorimetric methods, using the fluorescent ratiometric Ca2+ indicator Fura-2, the dye JC-1, and specific fluorogenic substrate, respectively. Our results indicated that treatment of HL-60 cells with H2O2 induced a transient increase in [Ca2+]c due to Ca2+ release from internal stores. The stimulatory effect of H2O2 on Ca2+ signal was followed by a mitochondrial membrane depolarization. Our results also indicated that H2O2 was able to increase the caspase-3 and -9 activities. The effect of H2O2 on caspase activation was time dependent, reaching a maximal caspase activity after 120 min of stimulation. Loading of cells with dimethyl BAPTA, an intracellular Ca2+ chelator, significantly reduced H2O2-induced mitochondrial depolarization and caspase activation. Similar results were obtained when the cells were pretreated with Ru360, a specific blocker of Ca2+ uptake into mitochondria. The findings suggest that H2O2-induced caspase-3 and -9 activation and mitochondrial membrane depolarization is dependent on rises in [Ca2+]c in human myeloid HL-60 cells.