We report the physiological role of OhrR as an organic peroxide sensor and transcription repressor in Xanthomonas campestris pv. phaseoli. In vivo exposure of X. campestris pv. phaseoli to either tert-butyl or cumene hydroperoxides efficiently neutralized OhrR repression of expression from the OhrR-regulated P1 promoter. H2O2 was a weak and non-physiological inducer of the system while other oxidants and metabolites of organic peroxide metabolism did not induce the expression from the P1. Northern blotting results indicated a correlation between concentrations of tert-butyl hydroperoxide used in the treatment and the induction of ohr (an OhrR-regulated gene) expression. In addition, the levels of ohr mRNA in cultures induced by various concentrations of tert-butyl hydroperoxide were reduced in cells with high levels of an organic peroxide metabolising enzyme (AhpC-AhpF) but not in cells with high catalase levels suggesting that organic peroxide interacts with OhrR. DNA band shift experiments using purified OhrR and the P1 promoter fragment showed that organic peroxide treatment prevented binding of the protein to the P1 promoter by oxidation of OhrR, as the inhibition of binding to the P1 promoter was reversed by addition of a reducing agent, DTT. The highly conserved cysteine residue C22 of OhrR is required for organic peroxide inducible gene expression. A mutant protein, OhrRC22S can repress the P1 promoter activity but is insensitive to organic peroxide treatment. Thus, OhrR is the first transcription repressor characterized that appeared to evolve to physiologically sense organic peroxides.