The sequential occurrence of plasma and mitochondrial membrane alterations, intra-cellular pH shifts and changes in intracellular Ca2+ concentration after induction of cell death was monitored by flow cytometry in Jurkat and HSB2-cells. Cell death was induced by treatment with anti-Fas antibodies or by irradiation. Phosphatidylserine (PS) exposure and plasma membrane integrity were measured with FITC-Annexin V adhesion and by Propidium Iodide exclusion. Transition of the mitochondrial membrane potential was monitored by the occurrence of decay of DiOC6 fluorescence. Intracellular pH shifts were monitored by changes in the ratio of fluorescence at 575 nm and at 635 nm of SNARF-1-AM. Fluctuations in intracellular Ca2+ concentration were established by changes in Fura red quenching. The Jurkat cells were sensitive to anti-Fas treatment, while HSB-2 cells were not. HSB-2 cells appeared more sensitive to radiation damage than Jurkat cells. In all experiments the transition of mitochondrial membrane potential occurred first, almost immediately followed by PS exposure. Fluctuations in intracellular Ca2+ concentration occurred later and were less outspoken. A decrease in intracellular pH occurred not earlier than 24 hours after anti-Fas treatment. Chelation of intracellular Ca2+ concentration with BAPTA-AM had no effect on the time sequence of cell death related events.