The gene for Agrobacterium tumefaciens OxyR, a peroxide sensor and transcriptional regulator, was characterized. Phylogenetic analysis of bacterial OxyR showed that the protein could be divided into four clades. The A. tumefaciens OxyR grouped in clade III that consists primarily of OxyRs of Alphaproteobacteria displayed the highest homology to OxyR from Rhizobium leguminosarum. oxyR is located next to, and is divergently transcribed from, a bifunctional catalase-peroxidase gene (katA). An A. tumefaciens oxyR mutant was constructed and shown to be hyper-sensitive to H2O2, but not to the superoxide generator, menadione, or an organic hydroperoxide. Exposure of A. tumefaciens to H2O2 resulted in induction of the catalase-peroxidase enzyme. This induction was abolished in the oxyR mutant. In vivo analysis of a katA::lacZ promoter fusion confirmed the results of enzyme assays and indicated that induction of the katA promoter by H2O2 was dependent on functional OxyR. We also examined the regulation of oxyR in A. tumefaciens. Exposure to H2O2 did not induce expression of the gene but simply changed OxyR from a reduced to an oxidized form. The in vivo oxyR promoter analysis showed that the promoter was auto-regulated and that transcription was not induced by H2O2.