Three thermophilic, anaerobic, strictly chemolithoautotrophic, sulphur- and/or thiosulphate-reducing bacteria, designated SL17T, SL19T and SL22T, were isolated from deep-sea hydrothermal samples collected at 13 °N (East Pacific Rise), Guaymas Basin (Gulf of California) and 23 °N (Mid-Atlantic Ridge), respectively. These strains differed in their morphology, temperature range and optimum for growth, energy substrates and 16S rRNA gene sequences. The G+C content of the genomic DNA was 41 mol% (SL22T), 42 mol% (SL17T) and 46 mol% (SL19T). Comparative analysis of phenotypic and phylogenetic traits indicated that strains SL17T and SL22T represented two novel species of the genus Desulfurobacterium and that strain SL19T should be considered as a novel species of the genus Thermovibrio. The names Desulfurobacterium pacificum sp. nov. (type strain SL17T=DSM 15522T=JCM 12127T), Desulfurobacterium atlanticum sp. nov. (type strain SL22T=DSM 15668T=JCM 12129T) and Thermovibrio guaymasensis sp. nov. (type strain SL19T=DSM 15521T=JCM 12128T) are proposed for these organisms. Furthermore, phylogenetic data based on 16S rRNA gene sequence analyses correlated with the significant phenotypic differences between members of the lineage encompassing the genera Desulfurobacterium, Thermovibrio and Balnearium and that of the families Aquificaceae and Hydrogenothermaceae. It is therefore proposed that this lineage represents a new family, Desulfurobacteriaceae fam. nov., within the order Aquificales.