Reactive oxygen species induce the expression of detoxification and repair genes critical for life in an aerobic environment. Bacterial factors that sense reactive oxygen species use either thiol-disulfide exchange reactions (OxyR, RsrA) or redox labile 2Fe–2S clusters (SoxR). We demonstrate that the reduced form of Bacillus subtilis OhrR binds cooperatively to two adjacent inverted repeat sequences in the ohrA control region and thereby represses transcription. In the presence of organic hydroperoxides, OhrR is inactivated by the reversible oxidation of a single conserved cysteine residue to the corresponding cysteine-sulfenic acid, and perhaps to higher oxidation states.