The energy released during the oxidation reactions in the mitochondrial respiratory chain is stored as an electrochemical potential consisting of a transmembrane electrical potential (Vmit) of about −180 mV and a proton gradient of about 1 pH unit; this drives the synthesis of the ATP required to fuel the cell’s energy-dependent processes. This key cellular system has been the subject of thousands of studies. However, data on how it is regulated by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have been precluded by their small size, their high motility, and the absence of appropriate methodologies.An approach toward a more quantitative assessment of Vmit within intact cells was based on the development of the potentiometric fluorescent dyes TMRE and TMRM, which rapidly equilibrate across membranes in accord with the Nernst equation.