Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural， low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE， a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent Km 227 ± 21 nM; Vmax 49 ± 21 pmol Ni(2+) min(-1) mg(-1) bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(ii) ions. This Ni(2+) transport is not inhibited by similar cations such as Mn(2+)， Zn(2+)， or Co(2+)， but is blocked by Cu(2+). Analysis of site-directed HupE mutants allowed the identification of several residues (H36， D42， H43， F69， E90， H130， and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein.