A novel aminopeptidase, termed aminopeptidase Y, was purified from yeast, Saccharomyces cerevisiae, as two molecular forms of 70 and 75 kDa, which were identical immunologically, catalytically and in N-terminal sequence up to 14 residues. They contained 0.81 and 0.84 mol of zinc atom/mol of protein, respectively. N-Gly-canase generated a single 53-kDa species from both forms, and endoglycosidase H gave a 54-kDa species. Immunoblotting after subcellular fractionation showed that aminopeptidase Y is localized in the vacuole/lysosome-soluble compartment as the 70-kDa form. Aminopeptidase Y hydrolyzed all amino acid-4-methylcoumaryl-7-amides (MCA) examined, especially Lys- and Arg-MCA. Dipeptidyl-MCAs were far more rapidly hydrolyzed (Lys-Ala-MCA/Lys-MCA = 350-fold, Arg-Arg-MCA/Arg-MCA = 150-fold). Hydrolysis of amino acid-MCAs or dipeptides was markedly enhanced by Co2+, whereas that of dipeptidyl-MCAs, tripeptides, and larger peptides was inhibited. Rabbit anti-aminopeptidase Y antiserum adsorbed over half the aminopeptidase activities in vacuolar extract from wild-type yeast cells. ABYS1 mutant cells from which the genes of four vacuolar proteases had been deleted contained aminopeptidase Y as a 74-kDa proform. This was converted to the mature form (70 kDa) by vacuolar extract of wild-type cells and by purified yeast vacuolar protease B.