Hydrogenase is the key enzyme responsible for H-2 production in dark fermentation. Therefore, the expression of hydrogenase gene may be a good indicator for the performance of a dark H-2 fermentation culture. In this study, we investigated the correlation between expression of the functional gene (hydA encoding for hydrogenase in Clostridium butyricum) and bioH(2) production activity during batch growth of an indigenous H-2-producing isolate C. butyricum CGS5 using sucrose as the sole carbon source. The copy number of hydA mRNA was determined by using reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR). The results show that the specific hydrogen production rate of C. butyricum CGS5 was essentially linearly proportional to the level of hydA expression (represented by the copy umber of hydA cDNA), whereas the profiles of microbial growth and volumetric H-2 Production rate followed a similar trend to that of the hydA DNA copies. (c) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.