The delta-subunit was isolated from the purified yeast F1. Partial protein sequences were determined by direct methods. From this information, degenerated primers were constructed. A part of the ATP delta gene was amplified by polymerase chain reaction from yeast genomic DNA. From the amplified DNA sequence, a nondegenerated oligonucleotide probe was constructed to isolate a 2.6-kbp BamHI-EcoRI DNA fragment bearing the whole gene. A 1036-bp DraI fragment was sequenced. A 480-bp open reading frame encoding a 160-amino-acid polypeptide is described. The deduced amino acid sequence is 22 amino acids longer than the mature protein, which is 138 amino acids long with a mass of 14,555 Da. The delta-subunit of Saccharomyces cerevisiae is 21%, 35%, 52% identical and 66%, 61% and 92% similar to the epsilon-subunit of Escherichia coli and the delta-subunits of beef heart and Neurospora crassa, respectively. A null mutant was constructed. The mutation was recessive and dramatically affected mitochondrial DNA stability since the transformed cells were 100% cytoplasmic petite. The double mutant (rho-, ATP delta::URA3) displayed low or no ATPase activity with an unstable catalytic sector, since a polyclonal antibody directed against the beta subunit did not coprecipitate the alpha subunit.