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Ultra-clean sampling methods and approaches typically used in pristine environments were applied to quantify concentrations of Hg species in water and microbial biomass from hot springs of Yellowstone National Park, features that are geologically enriched with Hg. Microbial populations of chemically-diverse hot springs were also characterized using modern methods in molecular biology as the initial step toward ongoing work linking Hg speciation with microbial processes. Molecular methods (amplification of environmental DNA using 16S rDNA primers, cloning, denatured gradient gel electrophoresis (DGGE) screening of clone libraries, and sequencing of representative clones) were used to examine the dominant members of microbial communities in hot springs. Total Hg (THg), monomethylated Hg (MeHg), pH, temperature, and other parameters influential to Hg speciation and microbial ecology are reported for hot springs water and associated microbial mats. Several hot springs indicate the presence of MeHg in microbial mats with concentrations ranging from 1 to 10 ng g 1 (dry weight). Concentrations of THg in mats ranged from 4.9 to 120,000 ng g 1 (dry weight). Combined data from surveys of geothermal water, lakes, and streams show that aqueous THg concentrations range from l to 600 ng L 1 . Species and concentrations of THg in mats and water vary significantly between hot springs, as do the microorganisms found at each site. � 2006 Published by Elsevier Ltd.