In Escherichia coli, the intrinsic levels of resistance to multiple antimicrobial agents are produced through expression of the three-component multidrug efflux system AcrAB-TolC. AcrB is a proton-motive-force-dependent transporter located in the inner membrane, and AcrA and TolC are accessory proteins located in the periplasm and the outer membrane, respectively. In this study, these three proteins were expressed separately, and the interactions between them were analyzed by chemical cross-linking in intact cells. We show that AcrA protein forms oligomers, most probably trimers. In this oligomeric form, AcrA interacts specifically with AcrB transporter independently of substrate and TolC.