Proteolytic activities were extracted from a dairy Lactobacillus helveticus strain and partially characterized. A first cell envelope proteinase (CEP) was extracted using a high ionic strength buffer, both in the presence and in the absence of Ca2+. Moreover, cell treatment by 5 M LiCl allowed for the selective removal of the S-layer protein and CEP, suggesting an enzyme ionic linkage to the cell envelope similar to that observed for the Slayer structure. The enzyme specificity against alpha(s1)-CN (f1-23) showed unusual activity on the Lys3-His4 bond compared with other proteinases of the same species. A second proteinase appeared to be linked to the cell membrane because it was extractable only after membrane disgregation by detergents. Its specificity against CN fractions and alpha(s1)-CN (f1-23) was different from that of the first CEP; moreover, the measured activity was lower than that of CEP.