Abstract The increasing rise in antibiotic resistance and the diminished discovery of new antimicrobials threatens global healthcare. Of particular concern are Gram-negative pathogens, organisms with an additional outer membrane (OM) that provides intrinsic resistance to multiple classes of antibiotics. Unlike the inner membrane (IM) that is composed solely of glycerophospholipids (GPLs), the OM is asymmetrical with GPLs found in the inner leaflet and lipopolysaccharide (LPS) localized to the outer leaflet. This unique membrane asymmetry affords protection from large polar molecules, as well as lipophilic compounds, creating an impervious barrier. Since the OM is essential, pathways required for its assembly are key targets for antimicrobial design. Currently, there are no antibiotics that directly target OM biogenesis in clinical use. Thus, it remains critical to investigate cell envelope biogenesis for future and current antimicrobial design. Over the last few decades, we have expanded our understanding of OM assembly revealing new targets for antimicrobial design. However, one major gap remained. How are GPLs transported from the IM to the OM across the aqueous periplasm? Recently, we discovered that key members of the AsmA-like family (YhdP, TamB, and YdbH) are critical for OM integrity and involved in GPL transport. We found that YhdP, TamB, and YdbH are redundant in their role in OM lipid homeostasis; however, they are not equivalent. Notably, all three proteins share homology and structural features with eukaryotic GPL transporters and are capable of spanning the periplasm. The overall objective of this application is to investigate the molecular mechanisms required for the assembly and maintenance of the Gram-negative OM. More specifically, we will characterize pathways required to transport GPLs across the cell envelope using E. coli as the model system. In the current application we will (i) characterize the major GPL transporters (YhdP, TamB, and YdbH), (ii) identify accessory proteins required for GPL transbilayer movement, and (iii) determine how loss of these systems impact OM lipid homeostasis and antibiotic resistance.