Arsenic oxidation is recognized as being mediated by both heterotrophic and chemoautotrophic microorganisms. Enrichment cultures were established to determine whether chemoautotrophic microorganisms capable of oxidizing arsenite As(III) to arsenate As(V) are present in selected contaminated but nonextreme environments. Three new organisms, designated as strains OL-1, S-1 and CL-3, were isolated and found to oxidize 10 mM arsenite to arsenate under aerobic conditions using CO2-bicarbonate (CO2/HCO3-) as a carbon source. Based on 16S rRNA gene sequence analyses, strain OL-1 was 99% most closely related to the genus Ancylobacter, strain S-1 was 99% related to Thiobacillus and strain CL-3 was 98% related to the genus Hydrogenophaga. The isolates are facultative autotrophs and growth of isolated strains on different inorganic electron donors other than arsenite showed that all three had a strong preference for several sulfur species, while CL-3 was also able to grow on ammonium and nitrite. The RuBisCO Type I (cbbL) gene was positively amplified and sequenced in strain CL-3, and the Type II (cbbM) gene was detected in strains OL-1 and S-1, supporting the autotrophic nature of the organisms.