A major tripartite multidrug efflux pump of Escherichia coli, AcrAB–TolC, confers resistance to a wide variety of compounds. The drug molecule is captured by AcrB probably from the periplasm or the periplasm/inner membrane interface, and is passed through AcrB and then TolC to the medium. Currently, there exist numerous crystallographic and mutation data concerning the regions of AcrB and its homologues that may interact with substrates. Starting with these data, we devised fluorescence assays in whole cells to determine the entire substrate path through AcrB. We tested 48 residues in AcrB along the predicted substrate path and 25 gave positive results, based on the covalent labelling of cysteine residues by a lipophilic dye-maleimide and the blocking of Nile red efflux by covalent labelling with bulky maleimide reagents. These residues are all located in the periplasmic domain, in regions we designate as the lower part of the large external cleft, the cleft itself, the crystallographically defined binding pocket, and the gate between the pocket and the funnel. Our observations suggest that the substrate is captured in the lower cleft region of AcrB, then transported through the binding pocket, the gate and finally to the AcrB funnel that connects AcrB to TolC.