e Arsenic and antimony are toxic metalloids and are considered priority environmental pollutants by the U.S. Environmental Protection Agency. Significant advances have been made in understanding microbe-arsenic interactions and how they influence arsenic redox speciation in the environment. However, even the most basic features of how and why a microorganism detects and reacts to antimony remain poorly understood. Previous work with Agrobacterium tumefaciens strain 5A concluded that oxidation of antimonite [Sb(III)] and arsenite [As(III)] required different biochemical pathways. Here, we show with in vivo experiments that a mutation in aioA [encoding the large subunit of As(III) oxidase] reduces the ability to oxidize Sb(III) by approximately one-third relative to the ability of the wild type. Further, in vitro studies with the purified As(III) oxidase from Rhizobium sp. strain NT-26 (AioA shares 94% amino acid sequence identity with AioA of A. tumefaciens) provide direct evidence of Sb(III) oxidation but also show a significantly decreased V max compared to that of As(III) oxidation. The aioBA genes encoding As(III) oxidase are induced by As(III) but not by Sb(III), whereas arsR gene expression is induced by both As(III) and Sb(III), suggesting that detection and transcriptional responses for As(III) and Sb(III) differ. While Sb(III) and As(III) are similar with respect to cellular extrusion (ArsB or Acr3) and interaction with ArsR, they differ in the regulatory mechanisms that control the expression of genes encoding the different Ars or Aio activities. In summary, this study documents an enzymatic basis for microbial Sb(III) oxidation, although additional Sb(III) oxidation activity also is apparent in this bacterium. T he metalloids arsenic (As) and antimony (Sb) are members of group 15 of the periodic table and are ubiquitous in the environment. Both are poisonous and have oxidation states of Ϫ3, 0, ϩ3, and ϩ5, with the last two being the most prevalent in the environment (1-5). The release of both As and Sb into the environment can occur either naturally or anthropogenically (e.g., mining), and both are considered by the U.S. Environmental Protection Agency to be priority environmental pollutants (6), with maximum drinking water standards of 10 ppb and 6 ppb for As and Sb, respectively (7). As has received more publicity due to As poisoning that has occurred and that continues (4, 8). However, Sb has emerged as a major contaminant in environments that contain mine tailings, such as those in China, Australia, New Zealand, and parts of Europe (for example, see references 5 and 9-11).Microorganisms are fundamental to elemental cycling in all environments, and this includes As (12, 13) and presumably Sb, although information for the latter is quite sparse. As cycling has been well documented and at present is thought primarily to involve arsenite [As(III)]Narsenate [As(V)] redox transformations and As methylation and demethylation reactions. As(V) is reduced for detoxification purposes (via ArsC) or respiratory...