The core of the vacuolar targeting signal of yeast carboxypeptidase Y (CPY) is recognized by the receptor Vps10p and consists of four contiguous amino acid residues, Gln24-Arg-Pro-Leu27, near the amino terminus of the propeptide (Valls, L.A., Winther, J. R., and Stevens, T. H. (1990) J. Cell Biol. 111, 361-368; Marcusson, E. G., Horazdovsky, B. F., Cereghino, J. L., Gharakhanian, E., and Emr, S. D. (1994) Cell 77, 579-586). In order to determine the sequence specificity of the interaction with the sorting receptor, substitutions were introduced into this part of the propeptide by semirandom site-directed mutagenesis. The efficiency of vacuolar sorting by the mutants was determined by immunoprecipitation of CPY from pulse-labeled cells. It was found that amino acid residues Gln24 and Leu27 were the most important ones. While it appears that Gln24 is essential for proper function, Leu27 can be exchanged with the other hydrophobic amino acid residues, isoleucine, valine, and phenylalanine. Tolerance toward various substitutions for Arg25 is fairly high, while substitution of Pro26 for uncharged amino acid residues also resulted in only weak missorting. In addition to the low requirement for sequence conservation, the position of the targeting element relative to the amino terminus of the propeptide was analyzed and found not to be critical.