The substrate specificity of the glucose-proton symport system was studied to gain information about the spatial relationship between the binding sites for glucose and proton. Charged glucose analogues such as amino sugars or sugar acids were not transported by the uptake system, with the exception of 2-amino-2-deoxy-D-glucose. This glucosamine was taken up in the charged form in uniport mechanism, i.e. without symport of proton. This result was interpreted to mean that the proton-binding site of the symport system is close to the hydroxyl at carbon 2 of glucose. This interpretation was strengthened by the following facts: The steric position of hydroxyl groups at carbons 1, 2 or 3 of glucose were especially important for efficient transport. O-Methylation was not tolerated at carbon 1, but it was tolerated at carbons 3, 4 or 6. The stoichiometric flow of proton and sugar could be disturbed by removal of hydroxyl group at carbon 1 of glucose. The pH-dependence of sugar transport is sugar-specific, e.g. the amino group at carbon 2 of glucose improves transport at higher pH. The configuration at carbon 2 of glucose influences the specificity for the symported ion. It is concluded that the coupled flow of proton and glucose occurs by simultaneous coordinate movement of both in a transmembrane channel.