INTRODUCTIONRhodamine 123 is a cationic fluorescent dye that is used to specifically label respiring mitochondria. The dye distributes according to the negative membrane potential across the mitochondrial inner membrane. Loss of potential will result in loss of the dye and, therefore, the fluorescence intensity. The dye has been used to monitor mitochondrial function in living cells. This protocol describes the labeling of mitochondria with Rhodamine 123.