Hydrogenase (Hyd) activity and H(2) production by Escherichia coli were studied at a low pH. H(2) production at pH 5.5 under glycerol fermentation was shown to be similar to 1.5-fold higher than that at pH 6.5 or above but less than that under glucose fermentation. It was inhibited by N,N'-dicyclohexylcarbodiimide: H(2) production inhibition was increased with decreasing pH and almost maximal inhibition was observed at pH 5.5. The data on H(2) production by single and double mutants with defects in different Hyd-enzymes and in fhlA gene suggest that under glycerol fermentation at a low pH, Hyd-1, Hyd-2 and Hyd-4 were operating in a reversed, non-H(2) producing mode. Moreover, a role of fhlA gene in Hyd-3 and Hyd-4 activity in H(2) production is proposed under glucose fermentation at a low pH. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.