Expression of the Escherichia coli uhpT gene, encoding the sugar phosphate transport protein, is induced by extracellular glucose-6-phosphate and requires the function of the uhpABC regulatory genes. The UhpA and UhpB proteins are related to the response-regulator and sensor-kinase proteins of two-component regulatory systems, whereas the UhpC protein is related to UhpT and homologous transport proteins. To investigate the role of segments of the membrane-associated UhpB and UhpC regulatory proteins, a series of mutations were constructed in vitro by insertion of a 12- or 24-bp oligonucleotide linker at 44 sites within the uhpABCT locus. The effect of these mutations on regulation of a uhpT-lacZ transcriptional reporter was assayed with the mutated uhp alleles in single copy on the chromosome. All but one of the insertions in uhpA or uhpT were inactive for transcription activation or transport, respectively. In contrast, about half of the insertions in uhpB and uhpC retained Uhp expression, and insertions at four sites in uhpB and at one site in uhpC conferred high-level constitutive expression. The constitutive mutants in UhpB resulted from insertions in the nonpolar amino-terminal half of the protein, and all insertions in that half of UhpB affected Uhp expression in some manner, which suggests that the transmembrane segments of UhpB might negatively regulate the kinase activity of the carboxyl portion. The constitutive behavior of all but one of these uhpB alleles was dependent on the presence of active forms of both UhpA and UhpC, which suggests that UhpB and UhpC act jointly as a complex in the signaling process.