It has recently been shown that the treatment of T. cruzi with cramoll 1,4, a seed lectin isolated from Cratylia mollis, induces an increase in cytoplasmic calcium concentration accompanied by the accumulation of calcium ions in the mitochondria, followed by an increase in the production of reactive oxygen species, a decrease in mitochondrial membrane potential and an absence of oxidative phosphorylation, leading to NCD with no DNA fragmentation. Given the rapid accumulation of calcium ions in the cytoplasm, the concomitant mitochondrial MG132 depolarization and the absence of DNA fragmentation observed here, the NCD observed in response to high doses of SBIs probably involves the accumulation of calcium ions in the mitochondrion, leading to the generation of ROS. These are the initial molecular steps leading to RMP and time-dependent cell lysis, the hallmarks of necrotic cell death. Furthermore, as EGTA did not interfere with cytoplasmic calcium overload, this ion must arise from intracellular pools, probably in the endoplasmic reticulum and/or acidocalcisomes. Recent studies of NCD in Dictyostelium have shown that mitochondrial uncoupling and ROS production are early events, occurring about 20 minutes after the induction of death and triggering the cascade of events involved in NCD. Mitochondrial changes can usually be reversed by removing the death-inducing factor. By contrast, lysosomal membrane permeabilization, which occurs after 70 to 100 minutes in Dictyostelium, is a ����point of no return���� event culminating in cell lysis after about 150 minutes of NCD activation. Thus, the correlation between RMP kinetics and commitment to cell death indicates that RMP represents the ����point of no return���� event in T. cruzi NCD. The extensive cellular degradation observed by microscopy is probably triggered by the release of reservosomal proteases. Recent TEM studies have described reservosome BKM120 rupture in response to trypanocidal drugs, but this is the first demonstration of the importance of RMP during T. cruzi cell death by complementary methods. It is not yet possible, from the results presented, to identify the intermediate steps leading to RMP, but the activation of a calpain-cathepsin cascade triggered by cytoplasmic calcium and/or direct oxidative damage may be crucial. The T. cruzi development stages residing in the mammalian host are the main targets of SBI treatment. Typical reservosomes storing material from endocytosis are visible only in epimastigote forms of T. cruzi, but all developmental stages present lysosome-related organelles and permeabilization of the reservosome membrane may play a crucial role in controlling cell death in mammalian stages of the parasite too. However, as amastigotes are 10 times more sensitive to SBIs than other stages, additional pathways may also contribute to cell death in these cells. The next step in our initial cellular and molecular characterization of the response of T. cruzi to SBIs will therefore involve the performance of these assays on amastigotes. Furthermore, given the limited therapeutic utility of the drug analyzed here, we will also test other SBI in future studies. Nevertheless, using classical SBIs acting on the epimastigote stage, we were able to obtain new insight into the response of T. cruzi to ergosterol synthesis inhibition. Based on the results of this work and those of published studies, we propose a model of T. cruzi necrotic cell death. The stress caused by the drugs first induces a rapid cytoplasmic calcium overload. The mitochondria concomitantly accumulate large amounts of calcium, impairing electron transport and leading to mitochondrial oxidative damage and inner membrane depolarization.