The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23 degrees 22' N, 44 degrees 57' W). The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20 degrees C. Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the Archaea or Bacteria domain and cloned. Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened. The majority of the archaeal sequences were affiliated with the Thermococcales (71%) and Archaeoglobales (22%) orders. A sequence belonging to the Thermoplasmales confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples. Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained. The majority of the bacterial sequences obtained were restricted to the Aquificales (18%), the epsilon subclass of the Proteobacteria (epsilon-Proteobacteria) (40%), and the genus Desulfurobacterium (25%). Most of the clones (28%) were confined to a monophyletic clade within the epsilon-Proteobacteria with no known close relatives. The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S(0), SO(4), thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents. The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid.