Mitochondrial membrane potential (Delta Psi(m)) plays an important role in cellular activity. Although Delta Psi(m) of intracellular mitochondria are relatively stable, the recent experiments with isolated mitochondria demonstrate that individual mitochondria show frequent fluctuations of Delta Psi(m). The current study is performed to investigate the factors that stabilize Delta Psi(m) in cells by observing Delta Psi(m) of individual isolated mitochondria with fluorescence microscopy. Here, we report that (1) the transient depolarizations are also induced for mitochondria in plasma membrane permeabilized cells, (2) almost all mitochondria isolated from porcine hearts show the transient depolarizations that is enhanced with the net efflux of protons from the matrix to the intermembrane space, and (3) ATP and ADP significantly inhibit the transient depolarizations by plural mechanisms. These results suggest that the suppression of acute alkalinization of the matrix together with the presence of ATP and ADP contributes to the stabilization of Delta Psi(m) in cells. (c) 2006 Elsevier Inc. All rights reserved.