The amino-terminal propeptide of carboxypeptidase Y (CPY) is necessary and sufficient for targeting this glycoprotein to the vacuole of Saccharomyces cerevisiae. A 16 amino acid stretch of the propeptide was subjected to region-directed mutagenesis using randomized oligonucleotides. Mutations altering any of four contiguous amino acids, Gln-Arg-Pro-Leu, resulted in secretion of the encoded CPY precursor (proCPY), demonstrating that these residues form the core of the vacuolar targeting signal. Cells that simultaneously synthesize both wild-type and sorting-defective forms of proCPY efficiently sort and deliver only the wild-type molecule to the vacuole. These results indicate that the PRC1 missorting mutations are cis-dominant, implying that the mutant forms of proCPY are secreted as a consequence of failing to interact with the sorting apparatus, rather than a general poisoning of the vacuolar protein targeting system.