Trithioarsenate is the predominant arsenic species at the source of alkaline, sulfidic geothermal springs in Yellowstone National Park. Kinetic studies along seven drainage channels showed that upon discharge the major initial reaction is rapid transformation to arsenite. When aerating a trithioarsenate solution in the laboratory, 10 to 20% of trithioarsenate dissociates abiotically before reaching a steady state with arsenite and thiosulfate. In the geothermal springs, trithioarsenate is completely converted to arsenite and rate constants of 0.2 to 1.9 min−1 are 40 to 500 times higher than in the laboratory, indicating microbial catalysis. Abiotic transformation of trithioarsenate to arsenate requires the presence of a strong oxidizing agent in the laboratory and no evidence was found for direct transformation of thioarsenates to arsenate in the geothermal drainage channels. The simultaneous increase of arsenite and arsenate observed upon trithioarsenate dissociation in some hot springs confirms that the main reactio is thioarsenate transformation to arsenite before microbially catalyzed oxidation to arsenate. In contrast to previous investigations in acidic hot springs, microbially catalyzed arsenate production in near-neutral to alkaline hot springs is not inhibited by the presence of sulfide. Phylogenetic analysis showed that arsenate production coincides with the temperature-dependent occurrence of organisms closely related to Thermocrinis ruber, a sulfur-oxidizing bacterium.