The amino acid sequence of the beta-subunit of bovine heart mitochondrial ATP synthase has been determined by protein sequence analysis. The polypeptide chain of 478 amino acids is blocked at its NH2 terminal. Comparison of this sequence with sequences of the corresponding proteins from Escherichia coli (Saraste, M., Gay, N.J., Eberle, A., Runswick, M.J., and Walker, J.E. (1981) Nucleic Acids Res. 9, 5287-5296) and maize and spinach chloroplasts Krebbers, E.T., Larrinua, I. M., McIntosh, L., and Bogorad, L. (1982) Nucleic Acids Res. 10, 4985-5002; Kurawski, G., Bottomley, W., and Whitfield, P.R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6260-6264) shows that the protein is highly conserved. 70% of residues are identical in E. coli and beef mitochondria. This contrasts with some of the other subunits in the enzyme complex which are much less conserved.