ABSTRACT Multidrug efflux transporters recognize a variety of structurally unrelated compounds for which the molecular basis is poorly understood. For the r esistance n odulation and cell d ivision (RND) inner membrane component AcrB of the AcrAB-TolC multidrug efflux system from Escherichia coli , drug binding occurs at the access and deep binding pockets. These two binding areas are separated by an 11-amino-acid-residue-containing switch loop whose conformational flexibility is speculated to be essential for drug binding and transport. A G616N substitution in the switch loop has a distinct and local effect on the orientation of the loop and on the ability to transport larger drugs. Here, we report a distinct phenotypical pattern of drug recognition and transport for the G616N variant, indicating that drug substrates with minimal projection areas of >70 Å 2 are less well transported than other substrates.