JC-1 (5,5',6,6'-tetrachloro1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide) dye has been used to monitor mitochondrial potential. The monomeric form has an emission maximum of ∼529 nm. The dye at higher concentrations or potentials forms red fluorescent J-aggregates with an emission maximum at 590 nm. The ratio of this green/red fluorescence is independent of mitochondrial shape, density, or size, but depends only on the membrane potential. It has been used to study whether all mitochondria in the same cell are at the same potential and whether membrane potential in a single long mitochondrion is uniform. JC-1 has also been useful in flow-cytometry studies, because the membrane potential can be followed without the need for confocal microscopy. This protocol describes the labeling of mitochondria in cultured cells with JC-1.