Although iron is an essential nutrient, its toxicity at high levels necessitates regulated transport. In Gram-negative bacteria a central target for regulation is the TonB protein, an energy transducer that couples the cytoplasmic membrane proton motive force to active transport of (FeIII)-siderophore complexes across the outer membrane. We have previously demonstrated the threefold repression of tonB transcription by excess iron in the presence of Fur repressor protein under aerobic conditions. In this report, we examine tonB regulation under anaerobic conditions where the solubility of iron is not a limiting factor and, presumably, siderophore-mediated transport is not required. Under these conditions, tonB transcription is repressed at least 10-fold by excess iron in the presence of Fur, but can be fully derepressed in the absence of Fur. Based on several lines of evidence, this anaerobic repression is not due to increased negative supercoiling as previously postulated. Our results rule out both supercoiling-mediated decreased promoter function and increased Fur binding as mediators of anaerobic repression. Under iron-limiting anaerobic conditions tonB expression is as high or higher than under iron-limiting aerobic conditions, suggesting that promoter function has not decreased anaerobically. Furthermore, under anaerobic conditions in tonB+ strains, tonB promoter function is insensitive to the gyrase inhibitor novobiocin and to changes in medium osmolarity and temperature, three conditions known to change levels of supercoiling. We also rule out effects of mutations in arcA or fnr as mediators of anaerobic repression. Results from in vivo dimethyl sulphate protection foot-printing indicate that Fur binds to an operator site between the -10 and -35 regions of the promoter, but not to a less homologous operator site centered at +26. The binding is, if anything, weaker under anaerobic conditions, indicating that anaerobic repression is not mediated through Fur. Additional changes in the in vivo footprint upstream from the promoter implicate a second factor in tonB anaerobic repression. Together, these results suggest that the mechanism responsible for this regulation (and, by analogy, that of other anaerobically repressed, iron-regulated genes such as cir, exbB, and fhuA) is a novel one.