The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca²⁺-overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca²⁺-permeable, non-selective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). This is followed by opening of large endogenous pores (COP) that allow molecules <800 Da to enter the cell. Lastly, the cells lyse, not by rupture of the plasmalemma, but rather through activation of a 'death' channel that lets large proteins (e.g. 140-160 kDa) leave the cell. These changes in permeability are accompanied by formation of membrane blebs. In this study, we took advantage of the well-known difference in affinities of various Ca²⁺-binding proteins for Ca²⁺ and Sr²⁺ versus Ba²⁺ to probe their involvement in each phase of the cell death cascade. Using fluorescence techniques at the cell population level (cuvette-based) and at the single cell level (time-lapse videomicroscopy) we found that replacement of Ca²⁺ with either Sr²⁺ or Ba²⁺ produced a delay in both the MTX-induced activation of COP, as indicated by the uptake of ethidium bromide, and on subsequent cell lysis, as indicated by the uptake of propidium iodide or the release of cell-associated GFP. MTX-induced responses were mimicked by ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single cell level revealed that Ba²⁺ not only delayed the time to cell lysis, but also caused a de-synchronization of the lytic phase. Lastly, membrane blebs, which in Ca²⁺-containing solutions were numerous and spherical, were poorly defined and greatly reduced in number in the presence of Ba²⁺. Taken together, these results suggest that intracellular high affinity Ca²⁺ binding proteins are involved in the MTX-induced changes in plasmalemmal permeability responsible for cell demise.