Escherichia coli produces molecular hydrogen (H-2) during glucose fermentation. This production of H-2 occurs via multiple and reversible membrane-associated hydrogenases (Hyd). Dependence of H-2 producing rate (VH2) by Hyd-4 (hyf) on glucose concentration was studied at different pHs. During growth on 0.2% glucose at pH 7.5 in JRG3615 (hyfA-B) and JRG3621 (hyfB-R) mutants (V-H2) was decreased similar to 6.7 and similar to 5 fold, respectively, compared to wild type. Only in JRG3621 mutant at pH 6.5 and 5.5 (V-H2) was severely decreased similar to 7.8 and similar to 3.8 fold, respectively. But when cells were grown on 0.8% glucose no difference between wild type and mutants was detected at any of the tested pHs. The results indicate Hyd-4 H-2 producing activity inhibition by high concentration of glucose mainly at pH 7.5. This is of significance to regulate Hyd activity and H-2 production by E. coli during fermentation. Copyright (c) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.