The lactose permease of Escherichia coli (LacY) is a highly dynamic membrane transport protein, while the Cys154→Gly mutant is crippled conformationally. The mutant binds sugar with high affinity, but catalyzes very little translocation across the membrane. In order to further investigate the defect in the mutant, fluorescent maleimides were used to examine the accessibility/reactivity of single-Cys LacY in right-side-out membrane vesicles. As shown previously, sugar binding induces an increase in reactivity of single-Cys replacements in the tightly packed periplasmic domain of wildtype LacY, while decreased reactivity is observed on the cytoplasmic side. Thus, the predominant population of wild-type LacY in the membrane is in an inward-facing conformation in the absence of sugar, sugar binding induces opening of a hydrophilic pathway on the periplasmic side, and the sugar-binding site is alternatively accessible to either side of the membrane. In striking contrast, the accessibility/reactivity of periplasmic Cys replacements in the Cys154→Gly background is very high in the absence of sugar, and sugar binding has little or no effect. The observations indicate that an open hydrophilic pathway is present on the periplasmic side of the Cys154→Gly mutant and that this pathway is unaffected by ligand binding, a conclusion consistent with findings obtained from single-molecule fluorescence and double electron-electron resonance.