The formate-hydrogen lyase (FHL) complex of Escherichia coli catalyzes the conversion of formate to hydrogen (H-2) and carbon dioxide (CO2) under anaerobic conditions in the absence of exogenous electron acceptors. The FHL complex consists of formate dehydrogenase (FdhH) and hydrogenase 3 (Hyd3) that are involved in a series of reactions, including formate oxidation (by FdhH), electron transport (by putative five small subunits of Hyd3) and proton reduction (by HycE, large subunit of Hyd3). In this study, the FHL gene cluster and iscR, a negative regulator of iron-sulfur cluster, of E. coli SH5 (BW25113 Delta hycA Delta hyaAB Delta hybBC Delta ldhA Delta frdAB) were altered to increase the FHL-dependent H-2 production activity. When FhlA, a transcriptional regulator of FHL, was overexpressed, the FHL-dependent H-2 production activity was improved significantly to 2.03 mu mol H-2 mg(-1) cdw min(-1) from 1.41 mu mol H-2 mg(-1) cdw min(-1). When an iscR deletion and/or fdhF overexpression was added, the whole-cell FHL activity was improved further to 2.80 mu mol H-2 mg(-1) cdw min(-1) (with Delta iscR) and 2.45 mu mol H-2 mg(-1) cdw min(-1) (with Delta iscR plus fdhF overexpression), respectively. The increase in whole-cell FHL activity was accompanied by an increase in the activity of its member enzymes, such as HycE and/or FdhH. In addition, the highly active recombinant strains exhibited stable performance during prolonged H-2 production with the repeated addition of formate. Overall, the FHL-dependent H-2 production activity of E. coli can be improved more than three-fold by modifying the expression of the relevant genes. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.