Monoclonal antibodies against peptidases of Lactococcus lactis were isolated and characterized: PEPN1–4 against a lysyl aminopeptidase PepN, PEPT1–5 against a tripeptidase PepT and PEPD1–3 against a dipeptidase PepD. These monoclonal antibodies reacted specifically with their respective antigens in crude cell extracts of Lc. lactis subspp. cremoris and lactis. A number of monoclonal antibodies cross reacted with proteins of other (lactic acid) bacteria. PEPT1, 2, 4 and 5 cross reacted weakly with a 35 kDa protein in Lactobacillus delbrueckii, while PEPT1 and PEPT2 reacted with proteins in the cell-free extract of Streptococcus thermophilus and Clostridium fervidus. Of the four isolated monoclonal antibodies against PepN, only PEPN3 cross reacted weakly with a 90 kDa protein in Escherichia coli cell-free extract, and the other three antibody species against PepN cross reacted with 80 kDa proteins of Lb. casei, Lb. delbrueckii, and Str. bovis, but not of Esch. coli. Of the three monoclonal antibodies against PepD, only PEPD1 and PEPD2 cross reacted with 40 kDa proteins of Lb. casei, Lb. delbrueckii and Str. bovis. All PEPN, PEPD and PEPT antibodies reacted with components in cell-free extracts of eleven different Lc. lactis strains, indicating that the peptidases of these strains were very similar to those of Lc. lactis subsp. cremoris WG2. However, Lc. lactis subsp. hordniae appeared to differ from the other Lc. lactis subspecies since only PEPT1, 2 and 5 reacted with a protein in the cell-free extract. Immunogold labelling of Lc. lactis WG2 with the isolated monoclonal antibodies revealed that PepN, PepD and PepT were located intracellularly. The intracellular location of these peptidases is discussed in relation to the supply of essential amino acids and peptides.