Geothermal springs within Yellowstone National Park (YNP) often contain arsenic (As) at concentrations of 10- 40 uM, levels that are considered toxic to many organisms. Arsenite (As(III)) is often the predominant valence state at the point of discharge but is rapidly oxidized to arsenate (As(V)) during transport in shallow surface water. The current study was designed to establish rates and possible mechanisms of As(III) oxidation and to characterize the geochemical environment associated with predominant microbial mats in a representative acid-sulfate-chloride (pH 3.1) thermal (58-62 °C) spring in Norris Basin, YNP. At the spring origin, total soluble As was predominantly As(III) at concentrations of 33 uM. No oxidation of As(III) was detected over the first 2.7 m downstream from the spring source, corresponding to an area dominated by a yellow filamentous S0-rich microbial mat. However, rapid oxidation of As(III) to As(V) was observed between 2.7 and 5.6 m, corresponding to termination of the S0-rich mats, decreases in dissolved sulfide, and commencement of a brown Fe/As-rich mat. Rates of As(III) oxidation were estimated, yielding an apparent first-order rate constant of 1.2 min-1 (half-life ) 0.58 min). The oxidation of As(III) was shown to require live organisms present just prior to and within the Fe/As-rich mat. Complementary analytical tools used to characterize the brown mat revealed an As:Fe molar ratio of 0.7 and suggested that this filamentous microbial mat contains iron(III) oxyhydroxide coprecipitated with As(V). Results from the current work are the first to provide a comprehensive characterization of microbially mediated As(III) oxidation and the geochemical environments associated with microbial mats in acidsulfate- chloride springs of YNP.