In this study with the model organism Agrobacterium tumefaciens, we used a combination of lacZ gene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of As(III) oxidation. A deletion mutation that removed the RpoN binding site from the aioBA promoter and an aacC3 (gentamicin resistance) cassette insertional inactivation of the rpoN coding region eliminated aioBA expression and As(III) oxidation, although rpoN expression was not related to cell exposure to As(III). Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters for aioB, phoB1, pstS1, dctA, glnA, glnB, and flgB that were examined by using qualitative RT-PCR and lacZ reporter fusions to assess the relative contribution of RpoN to their transcription. The expressions of aioB and dctA in the wild-type strain were considerably enhanced in cells exposed to As(III), and both genes were silent in the rpoN::aacC3 mutant regardless of As(III). The expression level of glnA was not influenced by As(III) but was reduced (but not silent) in the rpoN::aacC3 mutant and further reduced in the mutant under N starvation conditions. The rpoN::aacC3 mutation had no obvious effect on the expression of glnB, pstS1, phoB1, or flgB. These experiments provide definitive evidence to document the requirement of RpoN for As(III) oxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by As(III) or by this sigma factor.