The AcrB trimeric multidrug efflux transporter of Escherichia coli pumps out a very wide spectrum of compounds. Although minocycline and doxorubicin have been cocrystallized within the large binding pocket in the periplasmic domain of the binding protomer, nothing is known about the binding of many other ligands to this protein. We used computer docking to evaluate the interaction of about 30 compounds with the binding protomer and found that many of them are predicted to bind to a narrow groove at one end of the pocket whereas some others prefer to bind to a wide cave at the other end. Competition assays using nitrocefin efflux and covalent labeling of Phe615Cys mutant AcrB with fluorescein-5-maleimide showed that presumed groove-binders competed against each other, but cave-binders did not compete against groove-binders, although the number of compounds tested was limited. These results give us at least a hypothesis to be tested by more biochemical and genetic experiments in the future.