Biohydrogen has gained importance as an alternative energy source, and advances in molecular biology and biotechnology have raised the quality and efficiency of biohydrogen production from various microorganisms and substrates. Here, Escherichia coli proteins YdjA and YhjY have been identified as essential in biohydrogen production from glucose. The mutations ydjA and yhjY reduced biohydrogen productivity compared to the parent strain from 40 to 4 and 29 mu mol/mg protein, respectively. Through transcription analysis, it was determined that YdjA and YhjY are positive effectors of the FHL complex since their inactivation repressed fhlA. In addition, the FHL expression of the repressor gene, hycA, increased for the ydjA mutant, so YdjA reduces transcription of the HycA repressor. Hence, two new proteins have been identified that are important for biohydrogen production. Copyright (c) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.