Potassium ion (K(+)) plays vital roles in many aspects of cellular homeostasis including competing with sodium ion (Na(+)) during potassium starvation and salt stress. Therefore, one way to engineer plant cells with improved salt tolerance is to enhance K(+) uptake activity of the cells, while keeping Na(+) out during salt stress. Here, in search for Na(+)-insensitive transporter for this purpose, bacterial expression system was used to characterize two K(+) transporters, OsHAK2 and OsHAK5, isolated from rice (Oryza sativa cv. Nipponbare). The two OsHAK transporters are members of a KT/HAK/KUP transporter family, which is one of the major K(+) transporter families in bacteria, fungi and plants. When expressed in an Escherichia cola K(+) transport mutant strain LB2003, both OsHAK transporters rescued the growth defect in K(+)-limiting conditions by significantly increasing the K(+) content of the cells. Under the condition with a large amount of extracellular Na(+), we found that OsHAK5 functions as a Na(+)-insensitive K(+) transporter, while OsHAK2 is sensitive to extracellular Na(+) and exhibits higher Na(+) over K(+) transport activities. Moreover, constitutive expression of OsHAK5 in cultured-tobacco BY2 (Nicotiana tabacum cv. Bright Yellow 2) cells enhanced the accumulation of K(+) but not Na(+) in the cells during salt stress and conferred increased salt tolerance to the cells. Transient expression experiment indicated that OsHAK5 is localized to the plant plasma membrane. These results suggest that the plasma-membrane localized Na(+) insensitive K(+) transporters, similar to OsHAK5 identified here, could be used as a tool to enhance salt tolerance in plant cells. (C) 2010, The Society for Biotechnology, japan. All rights reserved.