This paper reports an investigation on the modulation of the mitochondrial permeability transition pore (MTP) by the membrane potential. Energized rat liver mitochondria loaded with a small Ca2+ pulse in sucrose medium supplemented with phosphate favor a high MTP "closed" probability because of the high membrane potential and therefore maintain a low permeability to sucrose. Upon depolarization by the addition of fully uncoupling concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) mitochondria favor a high MTP "open" probability and rapidly undergo a process of osmotic swelling following sucrose diffusion toward the matrix. A titration with FCCP reveals that discrete subpopulations of mitochondria with different gating potentials for MTP opening may exist, since increasing concentrations of FCCP increase the fraction of mitochondria undergoing osmotic swelling. We show that physiological effectors (Ca2+, Mg2+, ADP, palmitate) modify pore opening in a mitochondrial population by shifting the fraction of mitochondria with a functionally open pore at any given membrane potential. Many inducers and inhibitors may therefore affect the pore directly through an effect on the MTP voltage sensing rather than indirectly through an effect on the membrane potential. Thus, many effectors may induce pore opening by shifting the MTP gating potential to higher levels, whereas many inhibitors may induce pore closure by shifting the MTP gating potential to lower levels.