Enterobacter aerogenes HU-101, tested for its hydrogen production in batch cultures on various substrates, produced the highest amount of hydrogen when the substrate was glycerol. The yield of hydrogen is a function of the degree to which the substrates are reduced. To examine the effect of intracellular redox state on hydrogen yield, glucose-limiting chemostat cultures were carried out at various pH using strain HU-101 and its mutant AY-2. For both strains, the molar yield and the production rate of hydrogen and the hydrogenase activity in the cell-free extract were optimal at the culture pH of 6.3. The highest NADH/NAD ratio in both strains was also observed at pH 6.3, at which the ratio in AY-2 was more than two-fold that of HU-101. Furthermore, NAD(P)H-dependent hydrogen formation was observed in the cell-free extract of AY-2, and hydrogenase activity was found not in the cytoplasmic but in the cell membrane fraction, suggesting that a high intracellular redox state, that is a high NADH/NAD ratio, would accelerate hydrogen production by driving membrane-bound NAD(P)H-dependent hydrogenase. (C) 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.