The purpose of the research was to investigate the hydrogenase (hydA) gene and the corresponding transcript levels of Clostridium butyricum during continuous hydrogen fermentation. A quantitative real-time PCR (qrt-PCR) method was developed to specifically target the C. butyricum hydrogenase gene and mRNA in samples collected from a continuous, mixed-culture bioreactor over a period of 30 days (operation days 114-144). The detection limit of the qrt-PCR was 3.9 x 10(2) hydA copies and the linear range 3.9 x 10(2)-3.9 x 10(7) hydA copies. The results showed that after a re-inoculation of the bioreactor on day 120 the hydA copy numbers started to rise and stabilized after day 127. The number of hydA transcript continued to rise until day 142. The results demonstrate that this method is suitable for detecting the hydA gene and gene transcript levels of C. butyricum from bioreactor samples. The expression level of hydA gene changed during continuous operation and can, therefore, be a useful target for process performance monitoring. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.