A nuclear gene from Saccharomyces cerevisiae was cloned by genetic complementation of a temperature-sensitive respiratory-deficient mutant. DNA sequence analysis reveals that it encodes a protein with homology to Yme1, FtsH and Tma, proteins which belong to the AAA-protein family (ATPases associated with diverse cellular activities). The members of this family are involved in very different biological processes. Yme1p, a yeast mitochondrial protein, affects the rate of DNA escape from mitochondria to the nucleus and the Escherichia coli FtsH protein is apparently involved in the post-translational processing of PBP3, a protein necessary for septation during cell division. This newly sequenced gene, which we have designated AFG3 for ATPase family gene 3, encodes a putative mitochondrial protein of 760 amino acid residues that is closely related to FtsH, Tma (protein from Lactococcus lactis) and Yme1p with 58, 55 and 46% identity respectively.