ABSTRACT The OxyR transcription factor is a key regulator of the Escherichia coli response to oxidative stress. Previous studies showed that OxyR binding to a target promoter enhances RNA polymerase binding and vice versa, suggesting a direct interaction between OxyR and RNA polymerase. To identify the region of OxyR that might contact RNA polymerase, we carried out alanine scanning and random mutagenesis of oxyR . The combination of these approaches led to the identification of several mutants defective in the activation of an OxyR target gene. A subset of the mutations map to the DNA-binding domain, other mutations appear to affect dimerization of the regulatory domain, while another group is suggested to affect disulfide bond formation. The two mutations, D142A and R273H, giving the most dramatic phenotype are located in a patch on the surface of the oxidized OxyR protein and possibly define an activating region on OxyR.