The genus Hydrogenobacter consists of extremely thermophilic, obligately chemolithotrophic organisms that exhibit anaerobic anabolism but aerobic catabolism. Preliminary studies of the phylogenetic position of these organisms based on limited 16s ribosomal DNA sequence data suggested that they belong to one of the earliest branching orders of the Bacteria. In this study, the complete 16s ribosomal DNA sequences of two type strains, Hydrogenobacter thermophilus TK-6 and Calderobacterium hydrogenophilum 2-829, and another isolate, Hydrogenobacter sp. strain T3, were determined, and the phylogenetic positions of these organisms were examined. Our results revealed that the two type strains are members of a single genus, the genus Hydrogenobacter. Our results also verified the previous conclusion that the Aquifex-Hydrogenobacter complex belongs to a very early branching order, the "Aquificales." Within this order, the relationships among the various organisms are such that only a single family, the "AquiJcaceae," can be recognized at this time. Given the early branching point of the "Aqu$cales," the characteristics of these organisms support the view that the last common ancestor of existing life was thermophilic and suggest that this ancestor may have fixed carbon chemoautotrophically.It has been proposed that aerobic, thermophilic, hydrogenoxidizing, autotrophic bacteria played an important role in the primary productioln of organic matter on the early earth and that the ancestors of the Bacteria may have been thermophilic (4, 12). The increasing attention paid to thermophilic organisms as possible representatives of the earliest forms of life has resulted from the observation that the vast majority of the earliest branching organisms on 16s rRNA-based phylogenetic trees have this phenotype. The discovery of highly thermophilic, aerobic, hydrogen-oxidizing bacteria belonging to the genera Hydrogenobacter and Aquifex in geothermal hot springs was surprising, if one considers the hypothesis that free oxygen appeared only as a consequence of photosystem 11. It has been suggested that photosystem I1 developed much later in evolution, when the mean temperatures on the earth's surface were in the mesobiotic range (5). For this reason, the correct phylogenetic placement of these organisms deserves a thorough and detailed examination.Among the Bacteria, the Thermotoga lineage was previously thought to be the deepest phylogenetic branch and the most slowly evolving lineage (1,46,47). Interestingly, on the basis of its partial 16s rRNA sequence the more recently described highly t hermophilic hydrogen-oxidizing organism Aquifex pyrophilus, which was isolated from submarine hydrothermal vents, appeared to represent an even earlier branching event (12, 16). With an optimum growth temperature of 95"C, this organism is among the most thermophilic of the members of the Bacteria that have been described.On the basis of DNA-DNA hybridization data, the genus Aquifex was determined to be closely related to the genus Hydrogenobacter, ...