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Three thermophilic strains of chemolithoautotrophic Fe(III)-reducers were isolated from mixed sediment and water samples (JW/KA-1 and JW/KA-2T: Calcite Spring, Yellowstone N.P., WY, USA; JW/JH-Fiji-2: Savusavu, Vanu Levu, Fiji). All were Gram stain positive rods (∼0.5 × 1.8 μm). Cells occurred singly or in V-shaped pairs, and they formed long chains in complex media. All utilized H2 to reduce amorphous iron (III) oxide/hydroxide to magnetite at temperatures from 50 to 75°C (opt. ∼73°C). Growth occurred within the pH60C range of 6.5–8.5 (opt. pH60C 7.1–7.3). Magnetite production by resting cells occurred at pH60C 5.5–10.3 (opt. 7.3). The iron (III) reduction rate was 1.3 μmol Fe(II) produced × h-1 × ml−1 in a culture with 3 × 107 cells, one of the highest rates reported. In the presence or absence of H2, JW/KA-2T did not utilize CO. The G + C content of the genomic DNA of the type strain is 52.7 ± 0.3 mol%. Strains JW/KA-1 and JW/KA-2T each contain two different 16S rRNA gene sequences. The 16S rRNA gene sequences from JW/KA-1, JW/KA-2T, or JW/JH-Fiji-2 possessed >99% similarity to each other but also 99% similarity to the 16S rRNA gene sequence from the anaerobic, thermophilic, hydrogenogenic CO-oxidizing bacterium ‘ Carboxydothermus restrictus’ R1. DNA–DNA hybridization between strain JW/KA-2T and strain R1T yielded 35% similarity. Physiological characteristics and the 16S rRNA gene sequence analysis indicated that the strains represent two novel species and are placed into the novel genus Thermolithobacter within the phylum ‘ Firmicutes’. In addition, the levels of 16S rRNA gene sequence similarity between the lineage containing the Thermolithobacter and well-established members of the three existing classes of the ‘ Firmicutes’ is less than 85%. Therefore, Thermolithobacter is proposed to constitute the first genus within a novel class of the ‘ Firmicutes’, Thermolithobacteria. The Fe(III)-reducing Thermolithobacter ferrireducens gen. nov., sp. nov. is designated as the type species with strain JW/KA-2T (ATCC 700985T, DSM 13639T) as its type strain. Strain R1T is the type strain for the hydrogenogenic, CO-oxidizing Thermolithobacter carboxydivorans sp. nov. (DSM 7242T, VKM 2359T). [ABSTRACT FROM AUTHOR]