Expression of the Escherichia coli sugar phosphate transport system, encoded by the uhpT gene, is regulated by external glucose 6-phosphate through the action of three linked regulatory genes, uhpABC. The nucleotide sequence of the uhp region cloned from Salmonella typhimurium was determined. The deduced Uhp polypeptide sequences from the two organisms are highly related. Comparison with the corrected sequence from E. coli revealed that the four uhp genes are closely spaced, with minimal intergenic distances, and that uhpC is nearly identical in length to uhpT, both of which have substantial sequence relatedness along their entire lengths. To facilitate analysis of uhp gene function, we isolated insertions of a kanamycin resistance (Km) cassette throughout the uhp region. In-frame deletions that removed almost the entire coding region of individual or multiple uhp genes were generated by use of restriction sites at the ends of the Km cassette. The phenotypes of the Km insertions and the in-frame deletions confirmed that all three regulatory genes are required for Uhp function. Whereas the deletion of uhpA completely abolished the expression of a uhpT-lacZ reporter gene, the deletion of uhpB or uhpC resulted in a partially elevated basal level of expression that was not further inducible. These results indicated that UhpB and perhaps UhpC play both positive and negative roles in the control of uhpT transcription. Translational fusions of the uhpBCT genes to topological reporter gene phoA were generated by making use of restriction sites provided by the Km cassette or with transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with models predicting that UhpC and UhpT have identical transmembrane topologies, with 10 to 12 transmembrane segments, and that UhpB has 4 to 8 amino-terminal transmembrane segments that anchor the polar carboxyl-terminal half of the protein to the cytoplasmic side of the inner membrane.