An aspartic residue (Asp55) located in the putative transmembrane α-helix II of the melibiose( mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis. Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na + or H + coupled melibiose transport against a concentration gradient. ( 3H) p-nitrophenyl-α-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na + (or Li +) ions do no longer enhance the affinity of D55C permease for the co-transported sugar. In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose. These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na +-binding site and 2) that the cation and sugar binding sites may be overlapping.