Oxidation of glycolate to 2 CO2 and 3 H-2 (DELTAG-degrees' = +36 kJ/mol glycolate) by the proton-reducing, glycolate-fermenting partner bacterium of a syntrophic coculture (strain FlGlyM) depends on a low hydrogen partial pressure (p(H-2)). The first reaction, glycolate oxidation to glyoxylate (E-degrees' = -92 mV) with protons as electron acceptors (E-degrees' = -414 mV), is in equilibrium only at a p(H-2) of 1 muPa which cannot-be maintained by the syntrophic partner bacterium Methanospirillum hungatei; energy therefore needs to be spent to drive this reaction. Glycolate dehydrogenase activity (0.3-0.96 U . mg protein-1) was detected which reduced various artificial electron acceptors such as benzyl viologen, methylene blue, dichloroindophenol, K3[Fe(CN)6], and water-soluble quinones. Fractionation of crude cell extract of the glycolate-fermenting bacterium revealed that glycolate dehydrogenase, hydrogenase, and proton-translocating ATPase were membrane-bound. Menaquinones were found as potential electron carriers. Everted membrane vesicles of the glycolate-fermenting bacterium catalyzed ATP-dependent H-2 formation from glycolate (30-307 nmol H-2 . min-1 . mg protein-1). Protonophores, inhibitors of proton-translocating ATPase, and the quinone analog antimycin A inhibited H-2 formation from glycolate, indicating the involvement of proton-motive force to drive the endergonic oxidation of glycolate to glyoxylate with concomitant H-2 release. This is the first demonstration of a reversed electron transport in syntrophic interspecies hydrogen transfer.