Autotrophic carbon fixation was characterized in representative members of the three lineages of the bacterial phylum Aquificae. Enzyme activity measurements and the detection of key genes demonstrated that Aquificae use the reductive tricarboxylic acid (TCA) cycle for autotrophic CO(2) fixation. This is the first time that strains of the Hydrogenothermaceae and 'Desulfurobacteriaceae' have been investigated for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage could be identified within the Aquificae. Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and 'Desulfurobacteriaceae' use ATP citrate lyase. The first mechanism is likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest that ATP citrate lyase formed by a gene fusion of citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of this pathway. However, rather than having evolved within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase through lateral gene transfer. Aquificae play an important role in biogeochemical processes in a variety of high-temperature habitats. Thus, these findings substantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is widespread and contributes significantly to biomass production particularly in hydrothermal habitats.