The nucleotide sequence of the yeast nuclear AEP2 gene, required for the expression of the mitochondrial DNA-encoded subunit 9 of ATP synthase, predicts a primary translation product of 67.5 kDa. The ATP13 gene is allelic to AEP2 but was reported to encode a protein of about 42 kDa in size. We thus investigated genetically and biochemically the size of the AEP2 gene product. Genetic complementation assays using 3' truncated AEP2 genes, here shows that function is abolished by the removal of only 32 amino acids from the C-terminus of the predicted protein product. Cell-free translation of AEP2 produces a 64 kDa polypeptide (consistent with the AEP2 sequence) which is imported into mitochondria and processed to a 58 kDa product by the removal of a presequence of about 50 amino acids.