The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-m-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I Crenarchaeota and Group II Euryarchaeota, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic Methanococcales, as well as many uncultured Euryarchaeota, some of which have been identified in other vent environments. The particleattached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.Hydrothermal vent environments consist of multiple biotopes that span many of the physical and chemical conditions known to support life, including large ranges of temperature, pH, pressure, and oxygen concentrations and various forms of chemical energy, such as hydrogen sulfide, methane, and hydrogen (3,27,35). In all of these biotopes, microorganisms utilize either chemical energy derived from the reaction of water with rock at high temperatures or organic compounds derived from multiple sources (27). The microbial diversity varies from a single species of bacteria, as is the case for animal endosymbionts (52), to complex communities of different thermal groups of bacteria and archaea observed in hot vent fluids (42, 48, 49), sulfide structures (14,20,48,50), and microbial mats (31, 36). The subseafloor biotopes are probably the most enigmatic, since they are characterized by variable porosity (22) and complex thermal and chemical gradients (6) due to mixing of seawater with hydroth...