The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S]+ form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV- visible, EPR, NMR, and Mossbauer spectroscopies. The [4Fe-4S]+ form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S]2+, [3Fe- 4S](+/0), and to the stable [2Fe-2S]2+ form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Furthermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.