The hypothesis of an electric membrane potential generated by respiration or ATP hydrolysis in submitochondrial particles has been verified. To this end a number of synthetic ions penetrating lipid membranes were used.

Penetrating anions of phenyl dicarbaundecaborane (PCB⁻), tetraphenyl boron and picrate were shown to accumulate in sonicated submitochondrial particles in an energy-dependent manner. The process was inhibited by rotenone, antimycin and cyanide if supported by respiration, and by oligomycin, if ATP was used as the energy source. Uncouplers were inhibitory in both cases. The following oxidation reactions were found to support the energy-dependent accumulation of PCB⁻: oxidation of NADH by oxygen or fumarate; oxidation of succinate or ascorbate by oxygen; oxidation of NADPH by NAD⁺. In the latter case, which is the reverse of the energy-requiring transhydrogenase reaction, ion transport was inhibited by NADH and NADP⁺ as well as by uncouplers. Oxidation of NADH by NADP⁺ in the energy-requiring transhydrogenase reaction was accompanied by an efflux of PCB⁻ anions which had accumulated during succinate oxidation. The redox ‘succinate-ferricyanide’ couple could not be used as a supply of energy for the accumulation of PCB⁻

Particles deprived of the coupling factor F₁ showed a decreased ability for respiration-dependent anion uptake, the process being stimulated by oligomycin. ATP-driven PCB⁻ accumulation was completely absent in F₁-deprived particles but could be reconstituted after preincubation with F₁.

The active accumulation of anions penetrating into particles was readily distinguished from passive anion absorption, since the latter did not require energy and could be demonstrated both in native particles and in those deprived of F₁, as well as in phospholipid micelles. The energy-dependent accumulation of anions penetrating into submitochondrial particles was accompanied by alkalinization of the incubation medium. The efflux of ions upon the cessation of the energy supply induced acidification.

Anion accumulation was followed by the suppression of other energy-linked functions of submitochondrial particles. Under the same conditions the penetrating cations, dibenzyl dimethyl ammonium, tetrabutyl ammonium and triphenyl methyl phosphonium, did not affect either the pH of the medium or energy-linked functions.

It was concluded that a mechanism for ion accumulation in submitochondrial particles is specific for the sign of the charge but not for other features of the penetrating compounds. This mechanism operates in such a way that anions, but not cations, are pumped into the particle as if the process were supported by an electric field, orientated across the membrane, being positive inside the particles.