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Microbial metabolism of arsenic has gained considerable interest, due to the potential of microorganisms to drive arsenic cycling and significantly influence the geochemistry of naturally arsenic-rich or anthropogenically arsenic-polluted environments. Alvord Hot Spring in southeastern Oregon is a circumneutral hot spring with an average arsenic concentration of 4.5 mg L-1 (60 mu M). Hydrogeochemical analyses indicated significant arsenite oxidation, increased pH and decreased temperature along the stream channels flowing into Alvord Hot Spring. The dynamic range of pH and temperature over the length of three stream channels were 6.76-7.06 and 69.5-78.2 degrees C, respectively. Biofilm samples showed As(III) oxidation ex situ. 16S rRNA gene studies of sparse upstream biofilm indicated a dominance of bacteria related to Sulfurihydrogenibium, Thermus, and Thermocrinis. The lush downstream biofilm community included these same three groups but was more diverse with sequences related to uncultured OP10 bacterial phylum, uncultured Bacteroidetes, and an uncultured clade. Isolation of an arsenite oxidizer was conducted with artificial hot spring medium and yielded the isolate A03C, which is closely related to Thermus aquaticus based on 16S rRNA gene analysis. Thus, this study demonstrated the bacterial diversity along geochemical gradients of temperature, pH and As(III): As(V), and provided evidence of microbial arsenite oxidation within the Alvord Hot Spring system.