A total of 169 Lactobacillus strains from 12 species (Lb. acidophilus, Lb. brevis, Lb. buchneri, Lb. casei, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. delbrueckii, Lb. delbrueckii subsp. lactis, Lb. fermentum, Lb. helveticus, Lb. paracasei subsp. paracasei, Lb. plantarum and Lb. rhamnosus), isolated from raw milk and various milk products, and 9 Lactococcus lactis strains were evaluated for peptidase activities with five chromogenic substrates and a tryptic digest of casein. Within each species, the peptidase activity of the cell-free extracts of the strains varied. Furthermore, differences were observed between the Lactobacilhis species and Lc. lactis. Lb. helveticus had by far the highest hydrolysing activities towards all substrates, indicating the presence of powerful aminopeptidases, X-prolyl-dipeptidyl aminopeptidases and proline iminopeptidases. Lb. delbrueckii subsp. bulgaricus possessed high hydrolysing activities towards substrates containing proline, alanylprolyl–p–nitroanilide and prolyl–p–nitroanilide. On the other hand, Lb. fermentum and Lb. brevis could be considered as weakly proteolytic species. A more detailed study with highly proteolytic Lactobacillus strains indicated that at least three different proteinases or endopeptidases were present. Compared with Lc. lactis, the Lactobacillus strains had a much lower hydrolytic action on glutamyl-glutamic acid, suggesting that glutamyl aminopeptidase was absent in lactobacilli.