Recent results are in favour of a role for NFU-like proteins in Fe-S cluster biogenesis. These polypeptides share a conserved CXXC motif in their NFU domain. In the present study, we have characterized Arabidopsis thaliana NFU1-5 genes. AtNFU proteins are separated into two classes. NFU4 and NFU5 are part of the mitochondrial type, presenting a structural organization similar to Saccharomyces cerevisiae Nfu1p. These proteins complement a Delta isu1 Delta nfu1 yeast mutant and NFU4 mitochondrial localization was confirmed by green fluorescent protein fusion analysis. AtNFU1-3 represent a new class of NFU proteins, unique to plants. These polypeptides are made of two NFU domains, the second having lost its CXXC motif. AtNFU1-3 proteins are more related to Synechocystis PCC6803 NFU-like proteins and are localized to plastids when fused with the green fluorescent protein. NFU2 and/or NFU3 were detected in leaf chloroplasts by immunoblotting. NFU1 and NFU2 are functional NFU capable of restoring the growth of a Delta isu1 Delta nfu1 yeast mutant, when addressed to yeast mitochondria. Furthermore, NFU2 recombinant protein is capable of binding a labile 2Fe-2S cluster in vitro. These results demonstrate the presence of distinct NFU proteins in Arabidopsis mitochondria and plastids. Such results suggest the existence of two different Fe-S assembly machineries in plant cells.