The triplex efflux system AcrAB-TolC is a key player in multidrug resistance in E. coli. In this large protein complex, AcrB is the component that first takes up substrates from the periplasm and/or inner membrane of the cell. Many residues in AcrB have been found to make direct contact with substrate and line up the drug translocation pathway. For effective efflux, a substrate molecule has to bind and go through the drug translocation pathway in the periplasmic domain of AcrB. But how much does substrate binding and penetration rely on active efflux? To answer this question, we studied fluorescent labeling of sites lining up the substrate translocation pathway under three conditions devoid of active efflux. These conditions differ in the level of structure impairment in AcrB, and the observed level of labeling in response of structure changes differed for different sites. Labeling of 14 sites was examined in this study, and their data are summarized in Table 1. The most surprising discovery is the lack of significant response of the level of labeling to the absence of AcrA and TolC. In other words, substrate can bind and enter the translocation pathway of AcrB even without active drug efflux. As discussed above, the three subunits in an AcrB trimer adopt different conformations that are intrinsically not equally accessible by the substrates. The observation that all sites tested could be labeled to similar level as under the condition with active efflux seemed to suggest that although substrate were not extruded out of the cell, conversion between the three conformations was still possible. Since the proton relay pathway is intact, it is possible that translocation of protons could still occur, which drove the MK-4827 conformational rotation. Substrates could still migrate through the entire translocation pathway in AcrB and then be release back into the periplasm. The role of proton translocation in driving conformational rotation necessary for labeling was confirmed by the observation that the labeling of several sites was significantly weaker in AcrBD407A. These sites include S134C, N274C, D276C, R620C, and E673C. Since the D407A mutation has little effect on the overall structure of AcrB and does not disrupt its interaction with AcrA and TolC, it is reasonable to assume the observe decrease of labeling was a result of defect in proton translocation. We observed the most dramatic changes of labeling of the most sites when AcrB was dissociated into monomers. While for some residues including R717, T676, L668, F664, D566, and F666, labeling in CLAcrBDloop was close to the level of labeling in CLAcrB, for the rest of the sites tested labeling was much less in CLAcrBDloop. Sites labeled the least in CLAcrBDloop as compared to their levels of labeling in CLAcrB include Q89, N274, D276, and E673.