The binding and phosphorescence of Tb3+ in rat liver mitochondria and submitochondrial particles were investigated. Mitochondria were treated briefly with N-ethyl-maleimide (NEM) to prevent phosphate leak and Tb3+ chelation. Up to 30 nmol of Tb3+/mg of protein binds to mitochondrial membranes with high apparent affinity (Kd congruent to 6 microM). Generation of a membrane potential had no significant effect on the apparent affinity or capacity of Tb3+ binding in NEM-treated mitochondria. Mitochondrial bound Tb3+ phosphorescence can be induced selectively by excitation of aromatic amino acid residues. The decay of mitochondrial bound Tb3+ phosphorescence is biphasic. The phosphorescence of the slow phase (t1/2 = 0.45-0.70 ms) is quenched by monovalent salts, indicating a negative surface potential at low salt medium of -5.4 +/- 2.8 mV [10 mM 3-(N-morpholino)-propanesulfonic acid, pH 7.2, 5 microM Tb3+]. In submitochondrial particles, a surface potential of -6.5 +/- 2.7 mV was estimated under the same conditions. Energization did not affect the surface potential significantly in submitochondrial particles and only slightly in mitochondria. Analysis of the phosphorescence of mitochondrial bound Tb3+ reveals two binding sites with high (Kd = 1.5 microM) and low affinity (Kd = 29 microM). The high-affinity site is tentatively identified as the Ca2+ carrier. A fraction of the carrier-bound Tb3+ phosphorescence decays rapidly, presumably as a result of energy transfer to cytochromes in the membrane core. These intramembrane sites appear to move to the surface on the generation of a membrane potential. We conclude that the salt effect on the phosphorescence of the slow phase may serve as a reliable measure of delocalized surface potential in mitochondria and submitochondrial particles. Tb3+ binding to the high-affinity site may be useful as a probe for the mitochondrial Ca2+ translocator.