According to x-ray structure, the lactose permease (LacY) is a monomer organized into N- and C-terminal six-helix bundles that form a deep internal cavity open on the cytoplasmic side with a single sugar-binding site at the apex. The periplasmic side of the molecule is closed. During sugar/H(+) symport, a cavity facing the periplasmic side is thought to open with closure of the inward-facing cytoplasmic cavity so that the sugar-binding site is alternately accessible to either face of the membrane. Double electron-electron resonance (DEER) is used here to measure interhelical distance changes induced by sugar binding to LacY. Nitroxide-labeled paired-Cys replacements were constructed at the ends of transmembrane helices on the cytoplasmic or periplasmic sides of wild-type LacY and in the conformationally restricted mutant Cys-154-->Gly. Distances were then determined in the presence of galactosidic or nongalactosidic sugars. Strikingly, specific binding causes conformational rearrangement on both sides of the molecule. On the cytoplasmic side, each of six nitroxide-labeled pairs exhibits decreased interspin distances ranging from 4 to 21 A. Conversely, on the periplasmic side, each of three spin-labeled pairs shows increased distances ranging from 4 to 14 A. Thus, the inward-facing cytoplasmic cavity closes, and a cleft opens on the tightly packed periplasmic side. In the Cys-154-->Gly mutant, sugar-induced closing is observed on the cytoplasmic face, but little or no change occurs on periplasmic side. The DEER measurements in conjunction with molecular modeling based on the x-ray structure provide strong support for the alternative access model and reveal a structure for the outward-facing conformer of LacY.