The complete amino acid sequence of the mitochondrial ATPase inhibitor peptide was determined. The molecule contains 84 residues of which 40 are charged amino acids that occur in clusters along the chain. A section of the chain, located at the COOH-terminal end, contains several duplicated regions, the most prominent of which are pentapeptides. This section of the chain also contains all of the five histidines present in the molecule. Some of the physicochemical properties of the protein and an improved purification procedure are described.