The improvement in the resolution of confocal microscopy over conventional microscopy is comparable to that of magnetic resonance imaging over conventional radiography. Confocal microscopy is increasingly an essential analytical tool for studying the structure and physiology of living cells. The examples discussed in this chapter illustrate the use of confocal microscopy to image the intracellular distribution of pH, Ca2+, and electrical potential inside single living cells. Because many ion-indicating fluorophores distribute into both mitochondria and cytosol, ions can be measured individually in both compartments. By comparing mitochondrial and cytosolic pH and electrical potential, total mitochondrial proton-motive force can also be estimated. By using stable fluorophores and low levels of illumination, hundreds of images of living cells can be collected with negligible phototoxicity or photobleaching. Application of confocal microscopy promises to provide unique insights into mitochondrial function inside single living cells.