The production of a proteinase from Lactobacillus helveticus CRL 581 was studied, The highest specific activity was found at the early exponential growth phase of cells cultured in milk, The lowest levels of proteinase were detected in MRS broth, while in the casein-yeast extract-glucose broth enzyme production increased gradually during the fermentation and reached maximal values at the stationary phase, The proteinase, found to be associated with the cell membrane fraction, hydrolyzed p-casein more rapidly than a-casein. The enzyme was not released from washed cells in the presence or absence of calcium, which suggests that the enzyme did not undergo self-digestion.