We found new inhibitory function of lactoferrin and beta-casein in milk against cysteine proteases using reverse zymography. The inhibition of cathepsin L by lactoferrin was strongest and the inhibition kinetics were of a non-competitive type. Heat denatured lactoferrin lost the inhibitory activity completely, therefore the tertiary structure is essential to show the inhibition. Native lactoferrin was not degraded by papain during the assay condition. The intramolecular peptide, Y-679-K-695, of lactoferrin is an active domain and the synthesized peptide inhibited cysteine proteases. The Y-679-K-695 peptide showed 90% homology with the sequences of a common active site of cystatin family. beta-Casein and the active domain, synthesized L-133-Q(151), peptide inhibited cysteine proteases. Lactoferrin and beta-casein in milk might play a role in antiseptic and antiinfectious functions due to cysteine protease inhibition of bacteria and viruses. (C) 2003 Elsevier Science (USA). All rights reserved.