Lactococcus lactis ML3 possesses two different peptide transport systems of which the substrate size restriction and specificity have been determined. The first system is the earlier-described proton motive force-dependent di-tripeptide carrier (E. J. Smid, A. J. M. Driessen, and W. N. Konings, J. Bacteriol. 171:292-298, 1989). The second system is a metabolic energy-dependent oligopeptide transport system which transports peptides of four to at least six amino acid residues. The involvement of a specific oligopeptide transport system in the utilization of tetra-alanine and penta-alanine was established in a mutant of L. lactis MG1363 that was selected on the basis of resistance to toxic analogs of alanine and alanine-containing di- and tripeptides. This mutant is unable to transport alanine, dialanine, and trialanine but still shows uptake of tetra-alanine and penta-alanine. The oligopeptide transport system has a lower activity than the di-tripeptide transport system. Uptake of oligopeptides occurs in the absence of a proton motive force and is specifically inhibited by vanadate. The oligopeptide transport system is most likely driven by ATP or a related energy-rich, phosphorylated intermediate.