Anaerobically grown and glycolysing Escherichia coli produced H-2 and carried out H+-K+-exchange in two steps, the first of which had the fixed stoichiometry for DCCD-sensitive fluxes (2H(+)/K+), and the second one had a variable stoichiometry for DCCD-sensitive fluxes. H-2 production and the 2H(+)/K+-exchange were lost in the Delta fdhF or Delta hycA-H mutant. In the Delta fdhF mutant, H+-K+-exchange with K-m for K+-uptake of 2.3 mM and less K+-gradient between the cytoplasm and the medium were observed. H-2 production and H+-K+-exchange with a high K-m for K+-uptake were carried out in the uncD mutant; however, both H-2 production and H+-K+-exchange were lost in the Delta unc or uncE mutant. H-2 production was observed in the trkA trkD kdpA mutant. It was displayed in protoplasts with increased membrane permeability when donor or acceptor of reducing equivalents-formate with DTT or NADH respectively-was added. The F0F1 and the TrkA(H) or the F-0 and the TrkA(G) had been assumed to form the united supercomplexes, functioning as a H+-K+-pump or antiporter respectively (for review see Bioelectrochem Bioenerg 33:1, 1994). Results allow the proposal that H-2 production by FHL has a relationship with the H+-K+-exchange through a H+-K+-pump and via an H+-K+-antiporter, Formate and NADH can serve as a donor and an acceptor of reducing equivalent respectively, for operation of such supercomplexes.