The activity and the control of substrate-level phosphorylations in isolated yeast mitochondria were investigated. The oligomycin-insensitive ATP synthesis rate linked to 2-oxoglutarate oxidation is of similar order of magnitude to that observed in oxidative phosphorylation. The flux control coefficients of respiratory chain activity, translocase and phosphate carrier activities were close to zero. Kinetic control was confined to 2-oxoglutarate supply and 2-oxoglutarate dehydrogenase complex activity. The study of endogenous nucleotide phosphorylation showed that ADP is the first phosphate acceptor during this process. Moreover, the comparison between the whole intramitochondrial content of nucleotides and phosphate to the fraction involved in substrate-level phosphorylation indicated a metabolic compartmentation of nucleotides and phosphate. A linear relationship was observed between the 2-oxoglutarate-linked ATP synthesis rate and the internal phosphate potential: delta G'p = delta G'o + RT 1n ([ATP]/[ADP] [Pi]). The fact that the substrate-level phosphorylation process alone was able to maintain a high internal phosphate potential has an important bioenergetic consequence, particularly for yeast grown on fermentable substrates.