The nucleotide sequence of a chromosomal DNA fragment of Lactococcus lactis subsp. lactis SSL135, previously implicated in peptide utilization, has been determined. The genes oppDFBCA, encoding the oligopeptide transport system (Opp), and that encoding the endopeptidase PepO were located on this 8.9-kb DNA fragment. The oppDFBCA and pepO genes are probably organized in an operon. Analysis of the deduced amino acid sequences of the genes indicated that the oligopeptide transport system consists of two ATP-binding proteins OppD and OppF, two integral membrane proteins OppB and OppC, and a substrate-binding protein OppA. On the basis of the homology of OppF and OppD of L. lactis with other ABC (ATP-binding cassette) transporter proteins, the L. lactis Opp system can be classified as a member of this group. Two integration mutants, one defective in OppA and the other defective in PepO, were constructed. Growth of these mutants in a chemically defined medium with oligopeptides showed that the transport system, but not the endopeptidase, is essential for the utilization of peptides longer than three residues. Uptake of the pentapeptide Leu-enkephalin in glycolyzing lactococcal cells was followed by rapid hydrolysis of the peptide intracellularly. Importantly, extracellular hydrolysis of Leu-enkephalin is not observed. The OppA-deficient mutant was unable to transport Leu-enkephalin. Growth experiments with pasteurized milk revealed that transport of oligopeptides forms an essential part of the proteolytic system in lactococci.