Serine 130 is one of seven residues that form a total of seven hydrogen bonds with the sulfate completely sequestered deep in the cleft between the two lobes of the bilobate sulfate-binding protein from Salmonella typhimurium. This residue has been replaced with Cys, Ala, and Gly by site-directed mutagenesis in an Escherichia coli expression system. Replacement with the isosteric Cys caused a 3200-fold decrease in the sulfate-binding activity relative to the wild-type activity, whereas replacement with Ala and Gly resulted in only 100- and 15-fold decreases, respectively. The effect of the Cys substitution is attributed largely to steric effect, whereas the Gly substitution more nearly reflects the loss of one hydrogen bond to the bound sulfate with a strength of only 1.6 kilocalories per mole.