A yeast genomic DNA encoding a new vacuolar aminopeptidase, aminopeptidase Y, was isolated by using a cDNA fragment obtained by screening the lambda gt11 yeast cDNA library with anti-aminopeptidase Y antibody. The DNA sequence encodes 537 amino acids. The "mature" protein, whose NH2-terminal sequence was determined previously by analysis of the purified enzyme, consists of 481 amino acids, and the calculated molecular weight (52,900) coincides with the value obtained by SDS-polyacrylamide gel electrophoresis of the enzyme after removal of sugar chains, 53 kDa. The 56-residue preprosequence was divided into two parts by putative processing sites for signal peptidase and conversion to the mature form; the 21-residue presequence has a hydrophobic stretch which may function as the signal sequence for transit through the endoplasmic reticulum, and the 35-residue prosequence (4013 Da) accounts for the 4-kDa difference between proaminopeptidase Y in the vacuolar proteases-deleted ABYS1 mutant and wild-type mature enzyme. The aminopeptidase Y gene was localized on chromosome II by genetic mapping. A deletion mutant was constructed by disrupting the aminopeptidase Y gene. Vacuolar aminopeptidase activities toward Ala-4-methylcoumaryl-7-amide (MCA) and Lys-MCA were 13 and 20% of wild-type, and those in the presence of Co2+ were 2.2 and 2.8%, respectively. Mutant cells showed no ability to hydrolyze Lys-Ala-MCA to Lys and Ala-MCA, although vacuolar carboxypeptidase Y activity was similar to that in wild-type cells.