Flow cytometry combines the advantages of microscopy and biochemical analysis in a single highly sensitive technique for a rapid examination of numerous individual living cells. It has become a potent and essential tool in the studies of the physiology of the whole cell and its organelles. Rhodamine 123 is a vital fluorescent dye used in flow cytometry. As it is specifically concentrated in mitochondria because of the transmembrane potential that these organelles maintain in living cells, rhodamine 123 is thus a useful probe for monitoring the abundance and activity of mitochondria. A critical survey of the routine use of rhodamine 123 together with flow cytometry in mitochondrial research is presented.