The function of HKT1 in roots is controversial. We tackled this controversy by studying Na+ uptake in barley (Hordeum vulgare) roots, cloning the HvHKT1 gene, and expressing the HvHKT1 cDNA in yeast (Saccharomyces cerevisiae) cells. High-affinity Na+ uptake was not detected in plants growing at high K+ but appeared soon after exposing the plants to a K(+)-free medium. It was a uniport, insensitive to external K+ at the beginning of K+ starvation and inhibitable by K+ several hours later. The expression of HvHKT1 in yeast was Na+ (or K+) uniport, Na(+)-K+ symport, or a mix of both, depending on the construct from which the transporter was expressed. The Na+ uniport function was insensitive to external K+ and mimicked the Na+ uptake carried out by the roots at the beginning of K+ starvation. The K+ uniport function only took place in yeast cells that were completely K+ starved and disappeared when internal K+ increased, which makes it unlikely that HvHKT1 mediates K+ uptake in roots. Mutation of the first in-frame AUG codon of HvHKT1 to CUC changed the uniport function into symport. The expression of the symport from either mutants or constructs keeping the first in-frame AUG took place only in K(+)-starved cells, while the uniport was expressed in all conditions. We discuss here that the symport occurs only in heterologous expression. It is most likely related to the K+ inhibitable Na+ uptake process of roots that heterologous systems fail to reproduce.