The dependence of H-2 production on the formate channels, focA and FocB, by Escherichia coli at pH 5.5, 6.5 and 7.5 was shown using focA and focB mutants and comparing with the wild type. Moreover, effect of exogenous addition of formate (10 mM) on H-2 production was allotted. The results acquired propose that during glucose fermentation formate import can occur through FocB at different pHs; external formate drives PocA to import direction. However, during glycerol fermentation formate might be imported through FocB, whereas formate is exported preferentially through FocA at pH 7.5. Copyright (c) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.