Recently we identified a cytochrome c site that controls the binding of the protein to the mitochondrial mimetic membranes in a pH-dependent manner. This site (site L) encompasses Lys22, Lys25, His26, Lys27 and His33 residues that are arrayed in a confluent manner leading to the decrease of the pK_a of their lateral chain ionizable groups.  Thus, this site exhibits a pK_aobs around 7.0 and the lowering of the pH below this value favors its attachment to the acidic membranes. In this work, we tested, in cytochrome c-depleted rat liver mitoplasts, the effect of pH on the binding of exogenous cytochrome c to the inner mitochondrial membrane and the ability to restore the respiration and to produce ATP. The rate of oxygen consumption by cytochrome c-depleted mitoplasts and energized by succinate after the addition of 0.8 nmol horse heart cytochrome c, at pH 7.2, was 2.5 fold higher than the rate obtained at pH 6.2, suggesting at a first glance, an inhibitory effect of the acidic pH. However, the yield of ATP produced at pH 6.2 was 2.5 fold higher than the ATP yield at pH 7.2 and attained the ATP yield of whole mitochondria. Therefore, the decrease of the mitoplast respiration rate at pH 6.2 could be attributed to a higher DpH between the bulk medium and the mitochondrial matrix and suggests that at pH 7.2 mitoplasts behave as uncoupled mitochondria. The affinity of cytochrome c to the inner mitochondria membrane, at pH 7.2 and 6.2, was determined by measuring the respiration rate after the addition of different cytochrome c concentrations. The plot of respiration rate as a function of cytochrome c concentration revealed a hyperbolic quadratic profile. At pH 6.2, the saturation was attained with 0.1 nmol cytc/mg protein and at pH 7.2 this value was four fold higher. The double reciprocal plot of these data revealed two slopes for both investigated pH values indicating the existence of two cytochrome c populations. Over the highest cytochrome c concentration range, the pH decrease led to a decrease in the slope and pointed to an increase in the protein affinity to the inner mitochondrial membrane (Ks_app 45.7 nM at pH 6.2 and Ks_app 148.0 nM at 7.2). Over the lowest cytochrome c concentration range, the slopes were lower and identical in both pH values. Cytochrome c carbethoxylated at site L was unable to restore mitoplast respiration in both pH values. Thus, two cytochrome c populations could be functional in mitochondria. Supported by FAPESP, CNPq and FAEP-UMC.