In Escherichia coli, the UhpT transporter catalyzes the electroneutral accumulation of sugar 6-phosphate by exchange with internal inorganic phosphate (Pi). The substrate specificity of UhpT is regulated at least in part by constituents of an Asp388-Lys391 intrahelical salt bridge, and mutations that remove one but not both of these residues alter UhpT preference for organophosphate substrates. Using site-directed mutagenesis, we examined the role played by these two positions in the selection of the oxyanion countersubstrate. We show that derivatives having aliphatic or polar residues at positions 388 and 391 are gain-of-function mutants capable of transporting SO4 as well as Pi. These oxyanions share similar structures but differ significantly in the presence of a proton(s) on Pi. Our findings therefore lead us to suggest that the Asp388-Lys391 ion pair acts normally as a filter that prevents substrates lacking a proton that can be donated from occupying the UhpT active site.