The transcriptional repressor Fur binds to a 19-bp consensus sequence, 5'-GATAATGATAATCATTATC-3', under high iron conditions. The fepDGC-entS promoter of Escherichia coli contains two Fur-binding sites (FBS) offset by 6bp. Genetic studies of this promoter region revealed two mutations that exhibited a loss of iron regulation in vivo. One mutation altered the upstream portion of FBS 1, whereas the other, originally created to improve entS promoter strength, inadvertently altered the downstream portion of FBS 2. In both cases, there remains a 19-bp sequence that by current models should be sufficient for Fur binding. The effect of these mutations on Fur binding was examined using in vitro gel retardation assays and DNase I footprinting experiments. Though Fur bound wild-type DNA with high affinity, its affinity for the mutants was reduced, suggesting that both sites are required. In addition, gel shift studies demonstrated that the Fur-promoter complexes exhibit a unique hierarchy of binding, with distinct species forming at increasing concentrations of Fur. The DNA sequences bound in each gel-shifted species were determined using a coupled gel shift/footprint technique. The data presented here, with previously published data, suggest a new model for Fur-DNA interactions similar to that seen with the transcriptional repressor, DtxR. The model predicts that the 19-bp consensus Fur operator is configured as overlapping 13-mer sequences, and that two Fur dimers interact with these sequences from opposite faces of the helix.