Translocation of specific polypeptides across biological membranes during localization of protein is a highly conserved and essential process in all living cells. In Escherichia coli, the organism studied here, and in all the eubacteria, the general secretory, or Sec, system is the major route of export of proteins from the cytoplasm. The principles underlying the phenomenon of moving polypeptides across membranes are shared among diverse systems. The translocation channel itself, the heterotrimeric protein complex SecYEG, is highly conserved and has homologs in all three kingdoms of life, from single-cell organisms to mammals. The requirement that polypeptides be transferred in an unstructured state is common to protein localization in bacteria, in chloroplasts and in secretion through the endoplasmic reticulum. Initial transfer of a polypeptide into the channel appears to be mechanistically related whether it occurs co-translationally or after polypeptides are released from ribosomes. Structures show that if bound to ribosomes the cytoplasmic loops of SecY penetrate the ribosomal exit channel from which growing polypeptides emerge and if bound to SecA the same loops penetrate a deep cleft that is a binding site for the precursor ligand. What we learn from the research proposed is likely to provide insights into the molecular mechanism of other protein localization systems. We shall expand the number of precursors to be investigated from the two we have used in the past to more than 10. This will allow us to address functional and structural diversity We shall determine whether the ensemble of translocation complexes exists in a dynamic equilibrium or comprises subpopulations of fixed stoichiometry, each of which handles a subset of precursors. Studying the Sec system is a challenge because of the overlap of multiple levels of dynamic equilibria involved. Numerous components, each having multiple binding partners, interact at each stage of translocation starting with capture of an unfolded precursor by the chaperone SecB. SecA then joins to form a ternary complex, followed by passage of the precursor within the complex from SecB to SecA and subsequently on through the translocation channel. We aim to define changes within the complexes that allow transfer of precursor. The approach is based on our extensive understanding of interactions among the proteins involved. Another goal is to elucidate the mechanism of activation of SecYEG induced by co-assembly with SecA into liposomes. Powerful biophysical and biochemical ensemble approaches are combined with the single molecule technique, atomic force microscopy. Among the ensemble approaches used are titration calorimetry, EPR spectroscopy of spin-labeled proteins, analytical ultracentrifugation and column chromatography coupled to static light scatter. Additionally, we test translocation and coupled ATPase activity with a robust in vitro system. Our accumulated knowledge, well-defined model systems and past experience poise us to move the field toward a molecular description of the process of protein transport through membranes.