The subunit f of the yeast F1F0ATP synthase has been isolated from the purified enzyme. Amino acid composition, protein and peptide sequencing were performed. The data are in agreement with the sequence of the predicted product of the gene D9481.21 identified on the Saccharomyces cerevisiae chromosome IV. A 303-bp open reading frame encoding a 101-amino acid polypeptide is described. The deduced amino acid sequence from the ATP17 gene is 6 amino acids longer than the mature protein, which displays a molecular mass of 10567 Da. The protein is basic with a short hydrophobic segment located in the C-terminal part of the subunit. Subunit f remained associated with other F0 subunits upon sodium bromide treatment of the whole enzyme. A null mutant was constructed. The disrupted strain was unable to grow on glycerol medium and the mutation was recessive; rho- cells arose spontaneously. The null mutant mitochondria were devoid of oligomycin-sensitive ATPase, but still contained an active F1, while the subunits f, 6 and 8 were absent.