Proteolytic activity of native cysteine proteases was studied in bovine milk. Five fractions (fI-fV) with cysteine protease activity were separated from acid whey prepared from raw bovine milk by ion-exchange chromatography on Q-Sepharose. The hydrolytic action of the most active fractions (fIII and V), after further purification using gel permeation chromatography on Superdex S75, was studied against individual caseins. The two fractions contained different cysteine protease activities capable of hydrolyzing both alpha(s1)- and beta-casein. Studies of the effects of different reagents on the activity of partially purified fIII showed that the activity in this fraction was unaffected by aprotinin, slightly inhibited by p-chloromercuribenzoate and o-phenanthroline and completely inhibited by L-trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane, consistent with identification as a cysteine protease. Phenylmethylsulphonyl fluoride and pepstatin A reduced activity of fIII by 40% and 50%, respectively. The partially purified Ell retained similar to20% of its cysteine protease activity after heating at 55degreesC, 60degreesC, 65degreesC and 72degreesC for 40 min, 20 min, 10 min and 30s, respectively. Immunoblotting of fIII with antibodies to the bovine lysosomal cysteine protease, cathepsin B, clearly indicated the presence of immunoreactive cathepsin B in this fraction. This study presents strong evidence for the presence of a heterogeneous group of cysteine proteases in bovine milk, with one of these enzymes probably being cathepsin B. (C) 2002 Published by Elsevier Science Ltd.