The prokaryotic phylogenetic diversity was determined for a sample associated with an in situ growth chamber deployed for 5 days on a Mid-Atlantic Ridge hydrothermal vent (23 degrees 22'N, 44 degrees 57'W). The DNA was extracted from the sample and the 16S rDNA amplified by PCR. No Archaea were detected in the sample. Eighty-seven clones containing bacterial 16S rDNA inserts were selected. Based on restriction fragment length polymorphism analysis, 47 clones were unique, however, based on comparative sequence analysis some of these were very similar, and thus only 22 clones were selected for full sequence and phylogenetic analysis. The phylotypes were dominated by epsilon-Proteobacteria (66%). The remainder formed a novel lineage within the Proteobacteria (33%). One clone formed a distinct deeply branching lineage, and was a distant relative of the Aquificales. This report further expands the growing evidence that epsilon-Proteobacteria are important members in biogeochemical cycling at deep-sea hydrothermal ecosystems, participating as epibionts and free living bacteria.