In this report we examine the PEP-dependent phosphotransferase systems (PTSs) of Listeria monocytogenes EGD-e, especially those involved in glucose and cellobiose transport. This L. monocytogenes strain possesses in total 86 pts genes, encoding 29 complete PTSs for the transport of carbohydrates and sugar alcohols, and several single PTS components, possibly supporting transport of these compounds. By a systematic deletion analysis we identified the major PTSs involved in glucose, mannose and cellobiose transport, when L. monocytogenes grows in a defined minimal medium in the presence of these carbohydrates. Whereas all four PTS permeases belonging to the PTS(Man) family may be involved in mannose transport, only two of these (PTS(Man)-2 and PTS(Man)-3), and in addition at least one (PTS(Glc)-1) of the five PTS permeases belonging to the PTS(Glc) family, are able to transport glucose, albeit with different efficiencies. Cellobiose is transported mainly by one (PTS(Lac)-4) of the six members belonging to the PTS(Lac) family. In addition, PTS(Glc)-1 appears to be also able to transport cellobiose. The transcription of the operons encoding PTS(Man)-2 and PTS(Lac)-4 (but not that of the operon for PTS(Man)-3) is regulated by LevR-homologous PTS regulation domain (PRD) activators. Whereas the growth rate of the mutant lacking PTS(Man)-2, PTS(Man)-3 and PTS(Glc)-1 is drastically reduced (compared with the wild-type strain) in the presence of glucose, and that of the mutant lacking PTS(Lac)-4 and PTS(Glc)-1 in the presence of cellobiose, replication of both mutants within epithelial cells or macrophages is as efficient as that of the wild-type strain.