When cells of the anaerobe Streptococcus lactis 7962 are deprived of their normal fermentable energy source, active transport of galactosides is completely abolished although the membrane carriers are still capable of facilitating the equilibration of sugars across the cell membrane. In these nonmetabolizing cells it was possible to test the Mitchell hypothesis of obligatory coupling of proton movement with sugar transport. This hypothesis was supported by alkalinization of the medium observed when thiomethylgalactoside was added to a lightly buffered suspension of S. lactis cells. Conversely, addition of protons resulted in active transport of thiomethylgalactoside. Accumulation of thiomethylgalactoside to a concentration more than 20-times that in the external medium was induced by suddenly exposing cells to a medium at pH 6; no accumulation of thiomethylgalactoside was observed with cells exposed to pH 8.Active transport of thiomethylgalactoside occurred in the absence of energy metabolism when S. lactis cells were treated with valinomycin. This ionophore allowed intracellular K(+) to flow out, thus imposing a membrane potential (inside negative). This potential resulted in a proton uptake and an associated active transport of galactoside. The membrane potential was measured from the distribution ratio (inside/outside) of K(+) in the presence of valinomycin. The pH gradient was measured from the distribution ratio of [(14)C]methylamine. The protonmotive force, calculated from the membrane potential and the pH gradient, was found to be directly related to the accumulation of galactoside, in accordance with the chemiosmotic hypothesis.