INTRODUCTIONThe eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, it is important to study cellular organization and structure/function relationships. The mitochondrion is a double-membraned organelle that is important in various metabolic processes, including oxidative phosphorylation, electron transport, the Krebs cycle, β-oxidation of fatty acids, and part of the urea cycle. This protocol presents methods for labeling mitochondria in both live and fixed cells using several different fluorescent molecules. Rhodamine 123, tetramethylrhodamine ethyl ester (TMRE), and tetramethylrhodamine methyl ester (TMRM) are membrane-potential-sensitive, cationic fluorophores. Therefore, the mitochondrion must be functioning and generating a membrane potential in order to attract and maintain the dyes in the mitochondrion. These dyes are also used as sensitive indicators of the mitochondrial membrane potential. Rhodamine 123 is specific for the mitochondrion. In contrast, TMRM, TMRE, and the carbocyanine dyes can also label the endoplasmic reticulum to some degree. Mito Tracker CMTMRos has the added feature of chemical reactivity so that, once incorporated in the mitochondrion, it can chemically link to thiol groups and will not leave the mitochondrion when the membrane potential decreases as a result of fixation and/or cell death. Hence, it can be used to locate mitochondria in specimens that will subsequently be fixed. The effect of submicromolar concentrations of mitochondrial inhibitors, for example, KCN, rotenone, antimycin α, and uncouplers of oxidative phosphorylation, such as carbonylcyanide-m-chlorophenyl hydrazone (CCCP) and the related carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP), can be examined on isolated mitochondria or mitochondria in intact cells.