FNR, the transcriptional regulator of gene expression of anaerobic respiration in Escherichia coli, contains a cluster of cysteine residues at the amino terminus which resembles the metal-binding domains of metal-binding proteins. It is possible, therefore, (i) that FNR binds metals with the cysteines as ligands and (ii) that this property is related to the regulatory function of FNR. These questions were investigated, with the following results. Approximately 2.4 of the 4 cysteine residues of FNR can be alkylated with iodoacetate in permeabilized aerobic or anaerobic bacteria without the addition of reducing agents. The time required for half-maximal labelling of the cysteines was 50 min in anaerobic bacteria and 6 min in aerobic bacteria. The difference in the reactivity was specific for the cysteines of FNR. These cysteine residues were also highly reactive in anaerobically grown bacteria, when the growth medium contained chelating agents such as 1,10-phenanthroline (15 microM). The effect of the chelating agents was reversed by an excess of divalent metal ions such as Fe(II) or Cu(II) in the medium. The presence of 1,10-phenanthroline (10 microM) also inhibits the expression of fumarate reductase, an FNR-dependent enzyme. These results suggest that FNR exists in two different forms which differ in terms of the reactivity of their cysteine residues to iodoacetate. The interconversion of both forms appears to be regulated by the availability of O2 and by the binding of metal ions. The two forms of FNR may be involved in the regulation of O2-dependent gene expression.