AcrAB is a constitutively expressed, major multidrug efflux system of Escherichia coli. We have purified the cytoplasmic membrane component, AcrB, to near homogeneity, and reconstituted the protein into proteoliposomes. In the presence of ΔpH (outside acid), the protein catalyzed the extrusion of fluorescent phospholipids, which were then trapped by protein-free acceptor vesicles. Known substrates of AcrAB, such as bile acids, erythromycin, and cloxacillin, inhibited this activity. Addition of various drugs to AcrB-containing proteoliposomes, in the presence of ΔpH (inside acid) resulted in proton efflux, suggesting that AcrB is a proton antiporter. Interestingly, fluorescent lipid extrusion was accelerated strongly by the periplasmic protein AcrA in the presence of Mg²⁺, and at pH 5.0 AcrA alone produced a slow mixing of lipids of different vesicles, without causing the mixing of intravesicular material. These results suggest that AcrA brings two membranes together, and under certain conditions may even cause the fusion of at least the outer leaflets of the membranes, contributing to the ability of the AcrAB–TolC system to pump drugs out directly into the medium.