Mitochondrially mediated apoptosis is characterized by redistribution of proteins from mitochondria to cytoplasm following permeabilization of the outer mitochondrial membrane. We applied flow cytometry to quantify simultaneously the redistribution of two apoptogenic proteins, cytochrome c (cyt c) and Smac/DIABLO (Smac). Mammalian cells were treated with digitonin that selectively permeabilizes the plasma membrane. Following fixation, treated cells were infused successively with primary and secondary antibodies (the latter fluorescently tagged) enabling independent detection of cyt c and Smac. Digitonin-treated cells that retain cyt c or Smac in mitochondria generate strong fluorescence signals in flow cytometry. Cells in which cyt c or Smac have transited the outer mitochondrial membrane show greatly reduced fluorescence because the proteins are lost from the digitonin-permeabilized cells. Quantitative flow cytometry revealed that in 143B TK(-) cells treated with staurosporine, cyt c and Smac exit mitochondria asymmetrically, with cyt c redistribution preceding that of Smac. However, in HeLa cells likewise treated, cyt c and Smac exit mitochondria concurrently. Under other conditions of apoptotic induction, for example, 143B TK(-) cells treated with MT-21 (an apoptotic inducer that binds to the mitochondrial adenine nucleotide transporter), redistribution of Smac precedes that of cyt c. The various patterns of redistribution of these proteins were confirmed by immunocytochemical analysis and confocal microscopy. We conclude that flow cytometry can be employed effectively to quantify simultaneously the redistribution of cyt c and Smac from mitochondria to the cytosol. Moreover, differential redistribution of cyt c and Smac occurs under various conditions, thereby reflecting constraints on availability of these proteins to exit mitochondria after permeabilization of the outer membrane.