IscR is an iron-sulphur (Fe-S) cluster-containing transcription factor that represses transcription of the operon containing its own gene and the iscSUA-hscBA-fdx genes, whose products are involved in Fe-S cluster biogenesis. In this study, global transcriptional profiling of Escherichia coli IscR(+) and IscR(-) strains grown under aerobic and anaerobic conditions indicated that 40 genes in 20 predicted operons were regulated by IscR. DNase I footprinting and/or in vitro transcription reactions identified seven new promoters under direct IscR control. Among these were genes encoding known or proposed functions in Fe-S cluster biogenesis (sufABCDSE, yadR and yhgI) and Fe-S cluster-containing anaerobic respiratory enzymes (hyaABCDEF, hybOABCDEFG and napFDAGHBC). The finding that IscR repressed expression of the hyaA, hybO and napF promoters specifically under aerobic growth conditions suggests a new mechanism to explain their upregulation under anaerobic growth conditions. Phylogenetic footprinting of the DNase I protected regions of seven promoters implies that there are at least two different classes of IscR binding sites conserved among many bacteria. The findings presented here indicate a more general role of IscR in the regulation of Fe-S cluster biogenesis and that IscR contributes to the O(2) regulation of several promoters controlling the expression of anaerobic Fe-S proteins.