The Gram-negative bacterial cell envelope consists of an outer membrane and an inner membrane separated by an aqueous periplasm containing a thin peptidoglycan layer. In E. coli the inner membrane is a symmetrical phospholipid bilayer composed primarily of phosphatidylethanolamine (PE; 75%), phosphatidylglycerol (PG; 20%) and cardiolipin (CL; 5%). The inner membrane contains a-helical integral membrane proteins and lipoproteins anchored into its periplasmic side. The outer membrane has a more complex organization with lipopolysaccharide (LPS) and phospholipids, predominantly PE, PG and CL, forming an asymmetric bilayer containing integral b-barrel proteins and lipoproteins. These constituents work in concert to create a formidable barrier to antibiotics, detergents and other toxic chemicals. Each component of the outer membrane is synthesized in the cytoplasm and trafficked across the inner membrane and the periplasm before incorporation into the outer membrane. Over the last twenty years pathways have been identified for trafficking of LPS, integral b-barrel proteins and peripheral lipoproteins from the membranes. However, the enduring major conundrum in the field is the mechanism of phospholipid trafficking to the outer membrane; this pathway remains unknown. Using high through put genetic techniques (TraDIS) we will determine if specific pathways are required for lipid trafficking. Pathways identified through this approach will be studied further using biochemical and structural methods. Identification of such key pathways offers potential to develop new medicines to combat antimicrobial resistant microorganisms.