Escherichia coli is capable of producing hydrogen under anaerobic growth conditions. Formate is converted to hydrogen in the fermenting cell by the formate hydrogenlyase enzyme system. The specific hydrogen yield from glucose was improved by the modification of transcriptional regulators and metabolic enzymes involved in the dissimilation of pyruvate and formate. The engineered E. coli strains ZF1 (DeltafocA; disrupted in a formate transporter gene) and ZF3 (DeltanarL; disrupted in a global transcriptional regulator gene) produced 14.9, and 14.4 micromols of hydrogen/mg of dry cell weight, respectively, compared to 9.8 micromols of hydrogen/mg of dry cell weight generated by wild-type E. coli strain W3110. The molar yield of hydrogen for strain ZF3 was 0.96 mols of hydrogen/mol of glucose, compared to 0.54 mols of hydrogen/mol of glucose for the wild-type E. coli strain. The expression of the global transcriptional regulator protein FNR at levels above natural abundance had a synergistic effect on increasing the hydrogen yield in the DeltafocA genetic background. The modification of global transcriptional regulators to modulate the expression of multiple operons required for the biosynthesis of formate hydrogenlyase represents a practical approach to improve hydrogen production.