To gain a detailed understanding of the function of peptidases of Lactococcus lactis in growth in milk a set of 16 mutants was constructed lacking one or combinations of the following peptidases: PepO, PepN, PepC, PepT, PepXP. Growth experiments in milk showed that most of the mutants have a decreased growth rate, the general trend being that the more peptidases are inactivated the slower the mutant grows. A mutant lacking all five peptidases grows more than 10 times slower than the wild type. The activities of the cell envelope-associated proteinase (PrtP), of the oligopeptide transport system (Opp) and of peptidases still present in a mutant, were not influenced by its lack of certain peptidases or its decreased growth rate. Therefore the slow growth phenotype was directly caused by the absence of the peptidases and the consequent decreased capacity to release amino acids needed for growth. Furthermore, peptide catabolism was investigated in vivo using a chemically defined medium and a variety of peptides. The study presented shows for the first time that in milk at least PepO, PepN, PepC and PepT are directly involved in the release of amino acids needed for growth.