The glucose transporter of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar. It consists of a transmembrane subunit (IICBGlc) and a hydrophilic subunit (IIAGlc). The IICBGlc subunit consists of two domains. The NH2-terminal IIC domain (residues 1-386) spans the membrane eight times and contains the substrate binding site. The COOH-terminal hydrophilic IIB domain (residues 391-476) is accessible from the cytoplasmic side of the membrane. It contains the phosphorylation site (Cys421) and together with the IIC domain catalyzes the transfer of phosphoryl groups from the IIAGlc subunit to the transported solute. Starting from a plasmid vector containing ptsG under an inducible promoter, the IIB and the IIC domains have been subcloned separately, overexpressed in Escherichia coli, and purified by Ni2+ chelate affinity chromatography. Approximately 40 mg of IIBGlc-6H and 4 mg of IICGlc-6H could be purified from 1 liter of culture. Cells expressing IIBGlc-6H and IICGlc-6H separately have a three times longer generation time on glucose minimal medium than cells expressing wild-type IICBGlc. The rate of IIBGlc-6H phosphorylation determined in a nitrocellulose filter binding assay is indistinguishable from wild-type IICBGlc. The in vitro specific activity of IICGlc-6H in the presence of excess IIBGlc-6H is 2% of the control. IIBGlc-6H also complements the activity of a IICBGlc mutant with an inactive IIB domain (C421S) indicating that IIC and IIB are flexibly linked such that a free IIB domain can displace an inactive IIB domain from its contact site on the IIC domain. Based on this work, the secondary structure of the IIBGlc domain has been determined by isotope-edited NMR spectroscopy (Golic Grdadolnik, S., Eberstadt, M., Gemmecker, G., Kessler, H., Buhr, A., and Erni, B. (1994) Eur. J. Biochem. 219, 945-952).