The specificity of the S₁′ subsite of the proteolytic enzyme papain was investigated by studying the effect of l-α-amino acid amides on the enzyme-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester and by determining the kinetic parameters for the enzyme-catalysed hydrolysis of some N-benzyloxycarbonylglycyl-l-amino acid amides. These studies showed that the S₁′ subsite has a predilection for hydrophobic residues, in particular l-leucine and l-tryptophan. The specificity for these residues is manifest in both the binding and acylation steps. N-Benzyloxycarbonylglycine amide is not hydrolysed under comparable conditions, indicating that the amide group adjacent to and on the C-terminal side of the peptide bond about to be cleaved makes an important contribution to the rate of the papain-catalysed hydrolysis of peptides.