Molecular methods were employed to investigate the microbial community of a biofilm obtained from a thermophilic trickling biofilter reactor (TBR) that was operated long-term to produce H(2). Biomass concentration in the TBR gradually decreased as reactor bed height increased. Despite this difference in biomass concentration, samples from the bottom and middle of the TBR bed revealed similar microbial populations as determined by PCR-DGGE analysis of 16S rRNA genes. Nucleotide sequences of most DGGE bands were affiliated with the classes Clostridia and Bacilli in the phylum Firmicutes, and the most dominant bands showed a high sequence similarity to Thermoanaerobacterium thermosaccharolyticum.