Abstract Multidrug efflux pumps actively expel a wide range of toxic substrates across the cell envelope and play a significant role in both intrinsic and acquired multidrug resistance. Additionally, overproduction of efflux pumps is associated with virulence in many pathogenic bacteria. The AcrAB-TolC efflux pump is broadly conserved in gram-negative bacteria and transports many critical antibiotics used worldwide. We will investigate the assembly process in situ to understand multidrug efflux across two membranes in the context of the cell wall and cellular membrane environment. In the proposed approach, genetic, biochemical, and structural experiments will be used synergistically with new computational methods to characterize how efflux pump inhibitors and antibiotics impact efflux pumps in multidrug-resistant E. coli clinical isolates that overproduce AcrAB. These studies will generate a comprehensive picture of the tripartite pump assembly sequence, and the role of the cellular environment in the transport status of active drug efflux. We anticipate the resulting data will help elucidate the basis for drug resistance in bacterial pathogens and be an important step toward developing new strategies to block efflux.