Lipid II, the final precursor for peptidoglycan (PG) synthesis, consists of a disaccharide-pentapeptide moiety linked via a phosphodiester bond to a carrier lipid. Lipid II is assembled at the inner face of the cytoplasmic membrane and then flipped across the membrane to the periplasm, where the disaccharide-pentapeptide is incorporated into the existing PG sacculus by enzymes known as penicillin-binding proteins. The pathway for lipid II assembly and PG synthesis is generally well understood, with the important exception of how lipid II is flipped across the membrane. Genetic evidence and cell-based assays indicate that in E. coli the flippase is a membrane protein named MurJ, but biochemical evidence to support this hypothesis has been lacking {1}. Until now. This new report from Robinson’s group shows that purified MurJ binds lipid II with an apparent dissociation constant (Kd) of about 3µM. Under the same conditions, an alternative potential lipid II flippase, FtsW, had minimal affinity for lipid II. Follow-up experiments revealed binding of lipid II to MurJ is pH sensitive but indifferent to the concentration of sodium ions, suggesting a proton motive force rather than a sodium ion motive force powers transport across the cytoplasmic membrane. Interestingly, cardiolipin also bound to MurJ and interfered with binding of lipid II to MurJ, suggesting changes in cardiolipin levels might regulate MurJ activity. Collectively, these new findings confirm and extend the growing evidence that MurJ rather than FtsW or its homolog RodA is the lipid II flippase in E. coli.