Plant K(+) transporters of the HAK family belong to four rather divergent phylogenetic clusters, although most of the transporters belong to clusters I or II. A simple phylogenetic analysis of fungal and plant HAK transporters suggests that an original HAK gene duplicated even before fungi and plants diverged, generating transporters that at present fulfill different functions in the plant. The HvHAK1 transporter belongs to cluster I and mediates high-affinity K(+) uptake in barley roots, but no function is known for the cluster II transporter, HvHAK2, which is not functional in yeast. The function of HvHAK2 was investigated by constructing HvHAK1-HAK2 chimeric transporters, which were not functional even when they included only short fragments of HvHAK2. Then, amino acids characteristic of cluster II in the N terminus and in the first transmembrane domain were introduced into HvHAK1. All of these changes increased the Rb(+) K(m), introducing minimal changes in the Na(+) K(m), which suggested that HvHAK2 is a low-affinity, Na(+)-sensitive K(+) transporter. Using a K(+)-defective Escherichia coli mutant, we functionally expressed HvHAK2 and found that the predicted characteristics were correct, as well as discovering that the bacterial expression of HvHAK2 is functional at pH 5.5 but not at 7.5. We discuss whether HvHAK2 may be a tonoplast transporter effective for vacuolar K(+) depletion in K(+) starved plants.