Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H 2 O 2 . The present study shows that upon activation of death response by H 2 O 2 , a dose-and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca 2؉ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na ؉ and mitochondrial Ca 2؉ . Partial inhibition of cytosolic Ca 2؉ increase achieved by chelating extracellular or intracellular Ca 2؉ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca 2؉ is an additive result of release of Ca 2؉ from intracellular stores as well as by influx of extracellular Ca 2؉ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like Nacetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.Mitochondria are pivotal in controlling cell life and death (1). Maintenance of proper mitochondrial transmembrane potential (⌬ m ) 1 is essential for the survival of the cell as it drives the synthesis of ATP and maintains oxidative phosphorylation (2). Recently, the study of mitochondrial potential has become a focus of apoptosis regulation as many investigations demonstrate a major functional impact of mitochondrial alterations on apoptosis (2). Apoptosis is a process of cell death in which the cells undergo nuclear and cytoplasmic shrinkage; the chromatin is condensed and partitioned into multiple fragments, and finally the cells are broken into multiple membrane-bound bodies. In a number of experimental systems, disruption of ⌬ m constitutes a constant early event of the apoptotic process that precedes nuclear disintegration (3-5). For example, in thymocytes or tumor necrosis factor-stimulated U937 cells (3, 6), thymocytes or imexon-treated myeloma cells (5, 7), and PC-12 cells (8), a loss of ⌬ m occur as an early change associated with apoptosis. Lymphocytes with low ⌬ m show irreversible commitment to apoptosis in comparison to cells with high ⌬ m that do...