The mitochondrial ATP synthase is a membrane protein complex which couples the proton gradient across the mitochondrial inner membrane to the synthesis of ATP from ADP+Pi. The complex is composed of essential subunits for its motor functions and supernumerary subunits, the roles of which remain to be elucidated. Subunits g and A6L are supernumerary subunits, and the specific roles of these subunits are still matters of debate. To gain insight into the functions of these two subunits, we carried out the alignment and the homolog search of the protein sequences of the subunits and found the following features: Subunit g appears to have isoforms in animals, and the transmembrane domain of the animal subunit g contains a completely conserved acidic residue in the middle of a helix on the conserved side of the transmembrane helix. This finding implicates the conserved acidic residue as important for the function of subunit g. The alignment of A6L protein sequences shows a conserved aromatic residue at the N-terminal domain with which the N-terminal MPQL sequence comprises a unique MPQLX4Ar motif that can signify the protein A6L. The conserved aromatic residue may also be important for the function of A6L.