Investigation of the structure/function relationships of the sodium/glucose transporter (SGLT1) is crucial to understanding co-transporter mechanism. In the present study, we used cysteine scanning mutagenesis and chemical modification by methanethiosulphonate (MTS) derivatives to test whether predicted TM IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157 and D161) and two glucose/galactose malabsorption (GGM) missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to WT and was not studied further. Mutation of lysine 157 to cysteine (K157C) causes loss of phloridzin and MG binding (-methyl-D-glucopyranoside). These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulphonate hydrobromide (MTSEA). Mutation of threonine 156 to cysteine (T156C) reduces the affinity of aMG and phloridzin for T156C by ~5-fold and ~20-fold, respectively. In addition phloridzin protects cysteine 156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na⁺ currents.