A chimeric CaHAK1–LeHAK5 transporter with only 15 amino acids of CaHAK1 in the N-terminus mediates high-affinity K+ uptake in yeast cells. Kinetic and expression analyses strongly suggest that LeHAK5 mediates a significant proportion of the high-affinity K+ uptake shown by K+-starved tomato (Solanum lycopersicum) plants. The development of high-affinity K+ uptake, putatively mediated by LeHAK5, was correlated with increased LeHAK5 mRNA levels and a more negative electrical potential difference across the plasma membrane of root epidermal and cortical cells. However, this increase in high-affinity K+ uptake was not correlated with the root K+ content. Thus, (i) growth conditions that result in a hyperpolarized root plasma membrane potential, such as K+ starvation or growth in the presence of NH4+, but which do not decrease the K+ content, lead to increased LeHAK5 expression; (ii) the presence of NaCl in the growth solution, which prevents the hyperpolarization induced by K+ starvation, also prevents LeHAK5 expression. Moreover, once the gene is induced, depolarization of the plasma membrane potential then produces a decrease in the LeHAK5 mRNA. On the basis of these results, we propose that the plant membrane electrical potential plays a role in the regulation of the expression of this gene encoding a high-affinity K+ transporter.