A preparation that contained cytochrome c, mainly in the form of its ‘dimer’, was studied and compared with native cytochrome c with respect to its ability to support electron transfer and energy transformation in cytochrome c-depleted rat liver mitochondria. When the depleted mitochondria were titrated with either cytochrome c or the ‘dimer’, the extent of coupling between respiration and phosphorylation was enhanced, as manifested by an increase in the P/O ratio. The ‘dimer’ was relatively ineffective as an electron carrier in the respiratory system, but it was as effective as cytochrome c in reconstitution of oxidative phosphorylation in depleted mitochondria. Addition of ‘dimer’ to the depleted mitochondria, in the presence of a low, non-saturating concentration of cytochrome c, increased the P/O ratio without concomitant stimulation of respiration. Both cytochrome c and the ‘dimer’ stimulated spontaneous swelling and electron transport-driven proton translocation in depleted mitochondria. The pattern of action of cytochrome c and its ‘dimer’ is in accord with the assumption that they affect an early step in energy conservation.