The near ubiquitous distribution of nifH genes in sediments sampled from 14 high-temperature (48.0–89.0°C) and acidic (pH 1.90–5.02) geothermal springs in Yellowstone National Park suggested a role for the biological reduction of dinitrogen (N2) to ammonia (NH3) (e.g. nitrogen fixation or diazotrophy) in these environments. nifH genes from these environments formed three unique phylotypes that were distantly related to acidiphilic, mesophilic diazotrophs. Acetylene reduction assays and 15N2 tracer studies in microcosms containing sediments sampled from acidic and high-temperature environments where nifH genes were detected confirmed the potential for biological N2 reduction in these environments. Rates of acetylene reduction by sediment-associated populations were positively correlated with the concentration of NH4+, suggesting a potential relationship between NH4+ consumption and N2 fixation activity. Amendment of microcosms with NH4+ resulted in increased lag times in acetylene reduction assays. Manipulation of incubation temperature and pH in acetylene reduction assays indicated that diazotrophic populations are specifically adapted to local conditions. Incubation of sediments in the presence of a N2 headspace yielded a highly enriched culture containing a single nifH phylotype. This phylotype was detected in all 14 geothermal spring sediments examined and its abundance ranged from ∼780 to ∼6800 copies (g dry weight sediment)−1, suggesting that this organism may contribute N to the ecosystems. Collectively, these results for the first time demonstrate thermoacidiphilic N2 fixation in the natural environment and extend the upper temperature for biological N2 fixation in terrestrial systems.