Molecular hydrogen (H2) can be produced by Escherichia coli during mixed-acid fermentation of sugars (glucose) or glycerol via membrane-bound formate hydrogen lyase complex (FHL), which is composed by formate dehydrogenase H and hydrogenases (Hyd). This bacterium possesses different hydrogenases. pH can influence on activity of hydrogenases which might determine formate metabolism and H2 production.In this study it's shown that at slightly acidic pH (pH 6.5), during fermentation of glucose E. coli wild type produces H2. The H2 production yield and its rate determined by redox measurements was decreased in ΔfhlA mutant with deletion of transcriptional activator of Hyd-3 or Hyd-4, and stimulated in ΔhybB or ΔhybC mutants (with defective Hyd-1 or Hyd-2, respectively) and ΔhybC ΔhybB double mutant (with defective Hyd-1 and Hyd-2). H2 production rate was not inhibited in ΔhyfG mutant with defective Hyd-4. N,N’-dicyclohexylcarbodiimide (DCCD) or sodium azide, the inhibitors of proton translocating F0F1-ATPase, suppressed H2 production in wild type and mutant strains studied. Thus, at pH 6.5 Hyd-3 but not Hyd-4 may participate in the H2 formation whereas Hyd-1 and Hyd-2 probably operate in H2 oxidation mode. During glucose fermentation at pH 6.5 hydrogenases have relationship with the F0F1-ATPase. The relationship has been clearly demonstrated for the cells grown at alkaline pH (pH 7.5) that is playing an important role in generation of ΔμH+, or detoxification of formic acid formed during fermentation and neutralization of the cytoplasm.