Sequence analysis of the rpoN (2)- fixA intergenic region in the genome of Rhizobium etli CNPAF512 has uncovered three genes involved in nitrogen fixation, namely nifU, nifS and nifW. These genes are preceded by an ORF that is highly conserved among nitrogen-fixing bacteria. It encodes a putative gene product of 105 amino acids, belonging to the HesB-like protein family. A phylogenetic analysis of members of the HesB-like protein family showed that the R. etli HesB-like protein clusters with polypeptides encoded by ORFs situated upstream of the nifUS nitrogen fixation regions in the genomes of other diazotrophs. The R. etli ORF that encodes the HesB-like protein was designated iscN. iscN is co-transcribed with nifU and nifS, and is preferentially expressed under free-living microaerobic conditions and in bacteroids. Expression is regulated by the alternative sigma factor RpoN and the enchancer-binding protein NifA. A R. etli iscN mutant displays a reduction in nitrogen fixation capacity of 90% compared to the wild-type strain. This Nif(-) phenotype could be complemented by the introduction of intact copies of R. etli iscN.