Hydrothermal venting and the formation of carbonate chimneys in the Lost City hydrothermal field (LCHF) are driven predominantly by serpentinization reactions and cooling of mantle rocks, resulting in a highly reducing, high-pH environment with abundant dissolved hydrogen and methane. Phylogenetic and terminal restriction fragment length polymorphism analyses of 16S rRNA genes in fluids and carbonate material from this site indicate the presence of organisms similar to sulfur-oxidizing, sulfate-reducing, and methane-oxidizing Bacteria as well as methanogenic and anaerobic methane-oxidizing Archaea. The presence of these metabolic groups indicates that microbial cycling of sulfur and methane may be the dominant biogeochemical processes active within this ultramafic rock-hosted environment. 16S rRNA gene sequences grouping within the Methylobacter and Thiomicrospira clades were recovered from a chemically diverse suite of carbonate chimney and fluid samples. In contrast, 16S rRNA genes corresponding to the Lost City Methanosarcinales phylotype were found exclusively in high-temperature chimneys, while a phylotype of anaerobic methanotrophic Archaea (ANME-1) was restricted to lower-temperature, less vigorously venting sites. A hyperthermophilic habitat beneath the LCHF may be reflected by 16S rRNA gene sequences belonging to Thermococcales and uncultured Crenarchaeota identified in vent fluids. The finding of a diverse microbial ecosystem supported by the interaction of high-temperature, high-pH fluids resulting from serpentinization reactions in the subsurface provides insight into the biogeochemistry of what may be a pervasive process in ultramafic subseafloor environments.The Lost City hydrothermal field (LCHF) is located near the summit of the Atlantis Massif at a water depth of ϳ750 m (37, 38). Long-lived faulting and extensive uplift at the massif have resulted in the exposure of magnesium-rich, variably altered ultramafic rocks with lesser gabbroic material that is 1.5 to 2 million years of age. Fluid circulation within the massif is driven by serpentinization reactions and the cooling of the underlying mantle rocks. These reactions result in a combination of extreme conditions never before seen in the marine environment, which include venting of high-pH (from pH 9 to 11), 40 to 91°C hydrothermal fluids with high concentrations of dissolved hydrogen (H 2 ), methane (CH 4 ), and other lowmolecular-weight hydrocarbons (38). Mixing of the warm, high-pH fluids with seawater results in carbonate precipitation and growth of chimneys, which tower up to 60 m above the surrounding seafloor (38). Carbon-14 radioisotopic dating indicates that hydrothermal activity has been ongoing for at least 30,000 years (17). A large percentage of exposed seafloor on and near slow-and ultraslow-spreading ridges is likely to contain ultramafic rocks similar to those that host the LCHF (4, 12, 14). Therefore, this system offers a unique opportunity to study an ultramafic-rock-hosted submarine ecosystem that may be both widespread and ...