We have characterized the minimal functioning unit of UhpT, the secondary carrier that mediates exchange of phosphate and glucose 6-phosphate in Escherichia coli. Membranes of a UhpT overproducing strain were solubilized with 1.25% octyl beta-D-glucopyranoside, in the presence of 0.1% E. coli phospholipid and with 20% glycerol as the osmolyte stabilant. That soluble UhpT could bind its natural substrates was indicated by the protections afforded by sugar phosphates against thermal inactivation or chemical modification with pyridoxal 5'-phosphate. Moreover, the degree of protection correlated with the strength of interaction between UhpT and the test substrate (2-deoxyglucose 6-phosphate = glucose 6-phosphate greater than galactose 6-phosphate = glucose 1-phosphate much greater than glucose 6-sulfate). Other experiments demonstrated that soluble UhpT existed as a monomer. For example, during both high performance liquid chromatography and conventional gel permeation chromatography, the elution pattern of UhpT activity was measured directly by a rapid reconstitution technique. In both cases, and in the presence and absence of substrate, UhpT activity traveled as a single component of Mr 53,000, corresponding closely to the sequence prediction of 50,600. Finally, reconstitution was studied at protein to lipid ratios low enough to achieve between 0.075 and 1.5 UhpT monomers/proteoliposome. Specific activity was constant throughout this range, a finding consistent with the idea of a functional monomer. Mitochondria and chloroplasts provide the only other anion exchange carriers described at this level of biochemical resolution, and these organelle antiporters function as dimers. By contrast, work summarized here places their bacterial counterpart, UhpT, in the same class as the lactose carrier of E. coli and the glucose carrier of the human erythrocyte, both of which function as monomers. Consideration of this pattern in conjunction with the known hydropathy profiles of these proteins suggests a novel scheme for the classification of all secondary carriers, with implications for both the structure and origin of these transport proteins.