Our previous analysis of the transcriptome of Escherichia coli under nitrosative stress showed that the ytfE gene was one of the highest induced genes. Furthermore, the E. coli strain mutated on the ytfE gene was found to be more sensitive to nitric oxide than the wild-type strain. In the present work, we show that the mutation of the ytfE gene in E. coli yielded a strain that grows poorly under anaerobic respiratory conditions and that has an increased sensitivity to iron starvation. Furthermore, all examined iron-sulphur proteins have decreased activity levels in the strain lacking ytfE. Altogether, the results suggest a role for ytfE in iron-sulphur cluster biogenesis. YtfE was overexpressed in E. coli and it is shown to contain a di-iron centre of the histidine-carboxylate family.