To explain the limited nutritional value of milk cultured with proteinase-positive (Prt+) strains of Lactococcus lactis for the subsequent growth of dairy lactococci, we investigated further the time courses of modifications in the free amino acid and peptide contents of cultured milk. When growing in milk for up to 24 h, Prt+ strains of Lc. lactis progressively accumulated amino acids and casein-derived peptides. The growth of proteinase-negative (Prt−) wild-type strains and peptide transport mutants of Lc. lactis in cultured milk showed that casein-derived peptides could sustain growth up to 5×108 cfu/ml, depending on the extent of casein degradation during the preliminary growth of Prt+ strains and the Prt− strains. Of the casein-derived oligopeptides, <25% were transported into the cell and used for Lc. lactis growth. However, they played a prominent role, contributing 90% to growth. In contrast, di- and tripeptides did not contribute to growth, suggesting that either few were released from caseins or they did not supply essential amino acids.