Abstract An X-ray structure of the lactose permease of Escherichia coli (LacY) in an inward-facing conformation has been solved. LacY contains N- and C-terminal domains, each with six transmembrane helices, positioned pseudosymmetrically. Ligand is bound at the apex of a hydrophilic cavity in the approximate middle of the molecule. Residues involved in substrate binding and H⁺ translocation are aligned parallel to the membrane at the same level and may be exposed to a water-filled cavity in both the inward- and outward-facing conformations, thereby allowing both sugar and H⁺ release directly into either cavity. These structural features may explain why LacY catalyzes galactoside/H⁺ symport in both directions utilizing the same residues. A working model for the mechanism is presented that involves alternating access of both the sugar- and H⁺-binding sites to either side of the membrane.