The nrdDG promoter regulates transcriptional expression of the anaerobic ribonucleotide reductase of Escherichia coli, an essential enzyme required to supply the building blocks for DNA synthesis. In this work, binding of the pleiotropic FNR (fumarate and nitrate reduction) transcriptional regulator to the nrdDG promoter region and the effects of binding on transcription were investigated. Gel retardation analysis with purified FNR* demonstrated FNR interaction at two FNR sites, termed FNR-2 and FNR-1, while studies with altered FNR boxes indicated that the upstream FNR-2 site was essential for anaerobic activation of the nrdDG promoter. Although the FNR-1 site was not absolutely required, it allowed maximal expression of this promoter. These results suggest that the two sites have an additive effect in coordinating nrdDG expression in response to shifting oxygen concentrations.