Membrane proteins fulfill many critical functions in living cells, making them important targets for research and pharmaceutical development. However, the process by which these complex proteins are expressed and folded is not well understood. In E.coli, production of inner membrane proteins is a multi-step process involving a wide array of metabolic processes including transcription, translation, targeting of the ribosome to the membrane, insertion of the translated polypeptide, intramembrane folding and assembly, and the energy generating processes of central metabolism and oxidative phosphorylation. We have developed a synthetic, in vitro system that mimics all of these steps and efficiently produces inner membrane proteins in a folded and active form. Our cell-free production system contains a cell lysate, which provides the enzymatic machinery needed for protein synthesis, as well as vesicles derived from the inner membrane of E.coli, which provide membrane-bound chaperones and the lipid bilayer environment needed for folding and assembly of membrane proteins. Using this system, we have produced two membrane proteins, mannitol permease and the tetracycline pump, in high yields utilizing multiple turnovers of the required synthesis machinery. By taking advantage of the accessibility of our system, we have quantitatively determined how changes in signal recognition particle (SRP), SRP receptor, and vesicle concentrations affect overall production of active membrane proteins.