Variants of human stefin B were constructed by cassette mutagenesis. Val47 as the constituent of highly conserved QVVAG sequence was substituted by hydrophobic amino acids of increasing size - Ala, Ile and Phe. Recombinant proteins were expressed in E. coli and Ki values for papain were determined. Substitutions did not cause a major change in Ki value and we conclude that the interaction with the proteinases is not the reason for the conservation of the pentapeptide.