Oxidative stress plays a central role in the pathogenesis of atherosclerosis. By increasing the production of reactive oxygen species, such as H2O2, oxidative stress causes apoptosis of endothelial cells. Mechanisms by which H2O2 leads to apoptosis, however, are controversial. One study reported that Zn2+ released from H2O2 oxidation of metallothioneins is the cause of cell death [1], while the other suggests that H2O2 activates the TRPM2 channel, resulting in Ca2+ influx and cell death [2]. To address the controversy, we have examined the effect of H2O2 (1 mM, 6h) on human umbilical vascular endothelial cells using live cell imaging. We monitored changes in the intracellular distribution of free Ca2+ and Zn2+ using Fluo-4 and FluoZin-3 respectively. We stained lysosomes and mitochondria with LysoTracker and MitoTracker respectively, and dead cells with propidium iodide. Data recorded from N number of cells and n number of independent experiments are expressed as mean±SEM. P value of <0.05 (Student’s t-test) was considered statistically significant. We found that both Ca2+ and Zn2+ are highly enriched in lysosomes. Mitochondria showed little stain for either ion. H2O2 treatment increased the cytosolic levels of both ions in most cells. In some cells, however, we found striking redistribution of Zn2+ from lysosomes to mitochondria: There was a decrease in the number of lysosomes with Zn2+ (untreated: 61±6%, n/N=3/75; H2O2 treated: 21±1%, n/N=3/49; P<0.01), with a concomitant rise in mitochondria with Zn2+ (untreated: 11±1%, n/N=3/49; H2O2 treated: 41±4%, n/N=3/34; P<0.01). These effects were found in the absence of extracellular Zn2+, indicating redistribution of intracellular Zn2+. Inhibition of TRPM2 with PJ34 and 2-aminoethoxydiphenyl borate (2-APB) reduced the H2O2 induced release of Zn2+ into the cytoplasm (PJ34: 45±2%, n/N=3/43, P<0.01; 2-APB: 76±1 %, n/N=3/38, P<0.05), as well as its translocation from lysosomes to mitochondria (PJ34: 11±3%, n/N=3/46, P<0.05; 2-APB: 13±2%, n/N=3/35, P<0.05). Transfected HA-tagged TRPM2 channels showed co-localisation with the lysosomal markers, LysoTracker and CD63, suggesting TRPM2 mediates release of Zn2+ from lysosomes. We next determined the relevance of TRPM2 and Zn2+ redistribution to H2O2-induced endothelial cell death. Blockers of TRPM2 (PJ34 and 2-APB), as well as chelation of Zn2+ with TPEN (N,N,N’,N’-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine), completely inhibited H2O2-induced cell death, indicating TRPM2 mediated changes in the redistribution of Zn2+ contribute to endothelial cell death. In conclusion, we discovered a novel mechanism where H2O2 activation of TRPM2 causes a redistribution of Zn2+ from lysosomes to mitochondria and cytoplasm, resulting in endothelial cell death.