Glucose transport by the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) of listeria monocytogenes is inhibited by the bacteriocins, nisin, pediocin JD and leuconocin S. To investigate the mechanism of inhibition, PTS activity assays were performed with permeabilized, bacteriocin-treated L. monocytogens Scott A cells. In the presence of exogenous PEP, nisin stimulated the PTS while both pediocin JD and leuconocin S partially inhibited its activity. These results suggested that PTS enzymes were still active in bacteriocin-treated cells and the bacteriocin-induced PEP efflux may be a mechanism for inhibition of the PTS. To verify that PEP did efflux from bacteriocin-treated L. monocytogens Scott A cells, intracellular and extracellular PEP were measured by HPLC. All three bacteriocins induced efflux of PEP. Nisin, pediocin JD and leuconocin S also induced efflux of AMP, ADP and ATP. These studies indicate that bacteriocin inhibition of the glucose PEP:PTS in L. monocytogenes is due to efflux of intracellular metabolites, particularly