Background: The Resistance-Nodulation-Division (RND) family transporter AcrB plays a major role in the intrinsic and increased resistance of Escherichia coli to a large number of antibiotics. The distal binding pocket within this multidrug efflux transporter is very large, but the effort to define the roles of various residues facing this pocket through site-directed mutagenesis so far involved only the determination of minimal inhibitory concentrations of drugs in mutants. Methods: We measured in intact E. coli cells the kinetics of efflux of two substrates, nitrocefin (a cephalosporin) that is predicted mainly to bind to the upper, "groove" domain of the pocket, and L-alanyl-beta-naphthylamide (Ala-Naph) that is likely to bind to the lower, "cave" domain, in a number of site directed mutants of AcrB, where a hydrophobic or aromatic residue was changed into alanine. Results: The efflux of nitrocefin became attenuated by some mutations in the groove domain, such as I278A and F178A, but in some experiments a mutation in the cave domain, F628A produced a similar result. In some cases an increased value of KM was detected. The efflux of Ala-Naph was increased by mutations in the cave domain, such as F136A and I626A, but also by those in the groove domain (I277A, I278A, F178A). In most cases the increased V-max values appeared to be responsible. F610A mutation had a profound effect on the efflux of both substrates, as reported earlier. Conclusions: Our data show for the first time effects of various substrate-binding pocket mutations on the kinetics of efflux of two substrates by the AcrB pump. They also confirm interactions between substrates and drugs predicted by MD simulation studies, and also reveal areas that need future research. (C) 2016 Elsevier Inc. All rights reserved.