Chemical modification of Met-398 in carboxypeptidase Y from yeast has previously been shown to affect the specificity of the enzyme with respect to the P′₁ position. To confirm and further study the role of Met-398, this residue was substituted by a leucyl residue by means of site-directed mutagenesis. The mutagenesis was carried out in bacteriophage M13 on a subcloned fragment of PRC1, the structural gene for carboxypeptidase Y, using a dodecanucleotide containing the desired mutation as primer for secondary strand synthesis in vitro. A clone was identified in which codon 398 of the carboxypeptidase Y gene had been mutated from ATG to TTG while the rest of the subcloned region was conserved. This sequence was then reintroduced into the original PRC1 gene context and a ‡prc1 yeast strain was transformed with the resulting plasmid DNA. The mutant enzyme, Leu-398-carboxypeptidase Y, was isolated by affinity chromatography and shown to have the same molecular weight, N-terminal amino acid sequence and sugar content as carboxypeptidase Y. Cyanogen bromide degradation confirmed the absence of Met-398. Leu-398-CPD-Y was characterized kinetically using a series of N-blocked dipeptide and ester substrates with varying groups in the P′₁ position. Compared with the wild type enzyme Leu-398-carboxypeptidase Y showed an increased affinity towards substrates with bulky and hydrophobic groups in the P′₁ position. This is consistent with the leucyl side-chain being slightly smaller and more hydrophobic than the methionyl side-chain.