Site-directed sulfhydryl modification of transmembrane helix IX in the lactose permease of Escherichia coli was studied in right-side-out membrane vesicles with the thiol-specific reagents N-[14C]-ethylmaleimide (NEM) and methanethiosulfonate ethylsulfonate (MTSES) which are permeant and impermeant, respectively. Out of 20 mutants with a single Cys residue at each position in the helix, only five mutants label with NEM. (i) Cys residues at positions 291, 308, and 310 label at 25℃, and binding of substrate has no effect. (ii) Cys residues at positions 295 and 298 label only in the presence of substrate. NEM labeling at 0℃ indicates that alkylation of Cys residues at positions 295 and 308 is dependent on the thermal motion of the protein. In contrast, temperature has little effect on labeling of Cys residues at positions 291, 298, and 310. Interestingly, pretreatment with MTSES blocks NEM labeling of all the mutants. The findings demonstrate that the face of helix IX on which Arg302 is located is involved in ligand-induced conformational changes and accessible to water from the periplasmic surface of the membrane. Since Arg302 facilitates deprotonation of Glu325 (helix X) during turnover [Sahin-Toth, M., and Kaback, H. R. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6068-6073], the findings are consistent with the idea that this face of helix IX may comprise part of the H+ translocation pathway.