The fermentation process for hydrogen production has been widely reported. However, there is lack of information related to detailed kinetic studies. The aim of this work was to investigate biochemical kinetics of fermentative hydrogen production by a newly isolated strain of Clostridium butyricum W1. The research objectives were to clarify relationships between hydrogen fermentation and biochemical parameters and hydrogenases, and consequently to seek an index for hydrogen production. Time profiles of hydrogen production, cell growth, volatile fatty acid accumulation and [FeFe]hydrogenase expression level were described. The amount of hydrogen produced in a laboratory batch process was 45.45 mmol/L at 10 h and peak production rate was 7.61 mmol/l/h at 9 h. Cell growth rate peaked at 8 h. Lactic acid was a main by-product, followed by butyric acid and acetic acid. Quantification of [FeFe]hydrogenase mRNA was optimized by a real-time reverse transcriptase-PCR. Statistical analysis showed that [FeFe]hydrogen a se mRNA levels peak earlier than hydrogen production rate, and cell growth has a linear positive relationship with hydrogen production. Crown Copyright (c) 2008 Published by Elsevier Ltd on behalf of international Association for Hydrogen Energy. All rights reserved.