The nucleotide sequences of the glpT gene of Escherichia coli and its regulatory region have been elucidated and the primary structure of the glycerol-3-phosphate transport protein deduced. Extensive amino acid sequence homology was found with two other cytoplasmic membrane proteins: the functionally related hexose-6-phosphate transport protein, and the UHPC protein involved in regulating hexose-6-phosphate uptake. Although no significant amino acid sequence homology was found with other transport proteins, such as the arabinose, citrate, glucose, melibiose, lactose or xylose transporters, all of these proteins share a common secondary structure arrangement with the GLP T protein as they apparently contain twelve membrane-spanning alpha-helical segments. The promoter for glpT was located by transcript mapping and shown to overlap a site to which catabolite activator protein binds in vitro. These findings indicate how catabolite repression may be mediated but do not explain its physiological significance in glycerol metabolism.