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Reductive acetogenesis is an important metabolic process in the hindgut of wood-feeding termites. We analysed diversity and expression profiles of the bacterial fhs gene, a marker gene encoding a key enzyme of reductive acetogenesis, formyl tetrahydrofolate synthetase (FTHFS), to identify the active homoacetogenic populations in representatives of three different termite families. Clone libraries of polymerase chain reaction-amplified fhs genes from hindgut contents of Reticulitermes santonensis (Rhinotermitidae) and Cryptotermes secundus (Kalotermitidae) were compared with previously published fhs gene sequences obtained from Zootermopsis nevadensis (Termopsidae). Most of the clones clustered among the ‘Termite Treponemes’, which comprise also the fhs genes of the two strains of the homoacetogenic spirochaete Treponema primitia. The high abundance of treponemal fhs genes in all clone libraries was in agreement with the results of DNA-based terminal-restriction fragment length polymorphism (T-RFLP) analysis. Moreover, in mRNA-based T-RFLP profiles of the three termites, only expression of fhs genes of ‘Termite Treponemes’ was detected, albeit at different levels. In C. secundus, only one of the dominating phylotypes was transcribed, while in R. santonensis, the apparently less abundant fhs genes were the most actively expressed. Our results strongly support the hypothesis that spirochaetes are responsible for reductive acetogenesis in the hindgut of lower, wood-feeding termites.