INTRODUCTION Membrane-potential-dependent dyes such as Rhodamine 123, tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE) are useful as long as the mitochondrion maintains its negative membrane potential. MitoTracker is a commercially available fluorescent dye (Invitrogen/Molecular Probes) that, like the aforementioned dyes, labels mitochondria within live cells utilizing the mitochondrial membrane potential. However, MitoTracker is chemically reactive, linking to thiol groups in the mitochondria. The dye becomes permanently bound to the mitochondria, and thus remains after the cell dies or is fixed. In addition, it can be used in experiments in which multiple labeling diminishes mitochondrial function. This protocol describes the labeling of mitochondria in live and fixed cells with MitoTracker dyes.