A quantitative real-time PCR (qrt-PCR) method was developed for quantification of a hydrogen-producing Clostridium butyricum strain. The strain was the main hydrogen producer in a mixed culture bioreactor that was operated on a continuous-flow mode for 156 days. The qrt-PCR target was a segment of 16S rDNA, which was amplified from DNA extracted from bioreactor samples. The amplified target was quantified by measuring lanthanide fluorescence in a time-resolved manner. The linear range of this qrt-PCR assay was 10(2)-10(7) bacterial genomes. The results showed substantial variation (over two orders of magnitude) in the quantity of the target 16S rDNA of C. butyricum during the continuous-flow operation. Hydrogen production rate and butyrate concentration also varied with time. The method is applicable to any selected species in bioreactors and provides valuable insight into bioprocess performance and microbial community structure in complex dynamic systems. (C) 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.