The genome of Escherichia coli encodes two class I ribonucleotide reductases. The first, NrdAB, is a well-studied iron-dependent enzyme that is essential for aerobic growth. The second, NrdEF, is not functional under routine conditions, and its role is obscure. Recent studies demonstrated that NrdEF can be activated in vitro by manganese as well as iron. Since iron enzymes are potential targets for hydrogen peroxide, and since the nrdHIEF operon is induced during H2O2 stress, we hypothesized that H2O2 might inactivate NrdAB and that NrdEF might be induced to compensate. This idea was tested using E. coli mutants that are chronically stressed by H2O2. Contrary to expectation, NrdAB remained active. Its resistance to H2O2 depended upon YfaE, which helps to activate NrdB. The induction of NrdEF during H2O2 stress was mediated by the inactivation of Fur, an iron-dependent repressor. This regulatory arrangement implied that NrdEF has a physiological role during periods of iron starvation. Indeed, NrdEF supported cell replication in iron-depleted cells. Iron bound to NrdF when it was expressed in iron-rich cells, but NrdEF was functional only in cells that were both iron-depleted and manganese-rich. Thus NrdEF supports DNA replication when iron is unavailable to activate the housekeeping NrdAB enzyme.