The very early events of the intrinsic, damage-induced apoptotic pathway, i.e., upstream to Bax activation, probably consist of physico-chemical alterations (i.e., redox, pH or Ca2+ changes) rather then subtle molecular interactions, and in spite of many studies they remain unclear. One problem is that cells undergo apoptosis in an asynchronous way, leading to heterogeneity in the cell population that impairs the results of bulk analyses. In this study, we present a flow cytometric approach for studying Ca2+ alteration in apoptosis at the single cell level. By means of a multiparametric analysis, we could discriminate different sub-populations, i.e., viable and apoptotic cells and cells in secondary necrosis, and separately analyse static as well as dynamic Ca2+ parameters in each sub-population. With this approach, we have identified a set of sequential Ca2+ changes; two very early ones occur prior to any other apoptotic alterations, whereas a later change coincides with the appearance of apoptosis. Interestingly, the two pre-apoptotic changes occur simultaneously in all treated cells, i.e., at fixed times post-treatment, whereas the later one occurs at varying times, i.e., within a wide time range, concomitantly with the other apoptotic events.