The locations of the periplasmic proteins of Escherichia coli on standard two-dimensional gel patterns are described. The periplasmic fractions were prepared by osmotic shock of plasmolyzed whole cells and by release during EDTA-lysozyme treatment of whole cells. Within this fraction of proteins, we identify nine binding proteins (leucine-specific, glutamate-aspartate, glutamine, cystine, galactose, maltose, xylose, ribose, and arabinose) in addition to leucine-isoleucine-valine binding protein, which has been previously identified (Bloch, P. L., Phillips, T. A., and Neidhardt, F. C. (1980) J. Bacteriol. 141, 1409-1420). The identifications are based upon genetic criteria, protein induction, and comigration with purified protein. The levels of these proteins are compared in strains K12, B, and HA12 (a derivative of W). A technique was developed for renaturation of the ligand binding sites of periplasmic binding proteins in denaturing two-dimensional gels. This technique was used to demonstrate that leucine-specific and cystine binding proteins both have different isoelectric points in different strains. Renaturation was also used to demonstrate that there are two different charged forms for glutamine binding protein.