Hydrothermally influenced sediments from Middle Valley contain microorganisms, detectable microscopically and through phospholipid fatty acid analysis. Their abundances are estimated at about 105 cells/cm3 and are similar to other estimates of biomass in deep, hot environments. Agreement between direct counts and lipid analysis lends support to other studies of direct counts in deep marine sediments, and, in the future, lipid analyses could be used advantageously in such deep sediments, especially in conjunction with nucleic acid studies. The microbial community structure as seen from lipid biomarkers shows that the organisms that might otherwise be predicted as most abundant in these environments (high-temperature archaea, Desulfovibrio spp.) are undetectable. Communities in the five sediment samples are relatively similar to each other, except for changes in very long-chain fatty acids that increase with temperature. Otherwise the sediments do not show large or coordinated changes with temperature. Microbial communities in Middle Valley sediments may not have actively adapted to the changing environment downcore. Alternatively, as much remains to be discovered about microorganisms, active communities in these sediments at high temperature may represent new communities of high-temperature bacteria expressing very long-chain fatty acids.