When studied in vitro, mitochondrial permeability transition (MPT) is associated with an increase in mitochondrial permeability to solutes up to 1500 Da in mass and a loss of electrical potential difference across the inner mitochondrial membrane (Deltapsimit). The MPT has been implicated as being important in cellular calcium homeostasis, autophagy and cell death via necrosis and apoptosis. Thus, it is important to develop a valid technique for accurate measurement of this phenomenon in intact cells. We developed a procedure for the detection of MPT in intact cells that avoids the disadvantages associated with earlier approaches. In this new technique, unmodified (green-fluorescent) calcein is simultaneously introduced into the cytosol of millions of cells by the process of pinocytic loading and, to identify the position of individual mitochondria and to measure Deltapsimit, the cells are counter-stained with a red-fluorescing potentiometric dye. Using this approach with a variety of cell types, we demonstrate that cytosolic calcein is excluded from normal polarized mitochondria but enters them during MPT. This technique may be valuable in studies investigating the cellular functions of MPT.