The distribution of charged membrane-permeable molecular probes between intracellular organelles, the cytoplasm, and the outside medium is governed by the relative membrane electrical potentials of these regions through coupled equilibria described by the Nernst equation. A series of highly fluorescent cationic dyes of low membrane binding and toxicity (Ehrenberg, B., V. Montana, M.-D. Wei, J. P. Wuskell, and L. M. Loew, 1988. Biophys. J. 53:785-794) allows the monitoring of these equilibria through digital imaging video microscopy. We employ this combination of technologies to assess, simultaneously, the membrane potentials of cells and of their organelles in situ. We describe the methodology and optimal conditions for such measurements, and apply the technique to concomitantly follow, with good time resolution, the mitochondrial and plasma membrane potentials in several cultured cell lines. The time course of variations induced by chemical agents (ionophores, uncouplers, electron transport, and energy transfer inhibitors) in either or both these potentials is easily quantitated, and in accordance with mechanistic expectations. The methodology should therefore be applicable to the study of more subtle and specific, biologically induced potential changes in cells.