Plants can use ammonium (NH4+) as the sole nitrogen source, but at high NH4+ concentrations in the root medium, particularly in combination with a low availability of K+, plants suffer from NH4+ toxicity. To understand the role of K+ transporters and non-selective cation channels in K+/NH4+ interactions better, growth, NH4+ and K+ accumulation and the specific fluxes of NH4+, K+, and H+ were examined in roots of barley (Hordeum vulgare L.) and Arabidopsis seedlings. Net fluxes of K+ and NH4+ were negatively correlated, as were their tissue concentrations, suggesting that there is direct competition during uptake. Pharmacological treatments with the K+ transport inhibitors tetraethyl ammonium (TEA+) and gadolinium (Gd3+) reduced NH4+ influx, and the addition of TEA+ alleviated the NH4+-induced depression of root growth in germinating Arabidopsis plants. Screening of a barley root cDNA library in a yeast mutant lacking all NH4+ and K+ uptake proteins through the deletion of MEP1-3 and TRK1 and TRK2 resulted in the cloning of the barley K+ transporter HvHKT2;1. Further analysis in yeast suggested that HvHKT2;1, AtAKT1, and AtHAK5 transported NH4+, and that K+ supplied at increasing concentrations competed with this NH4+ transport. On the other hand, uptake of K+ by AtHAK5, and to a lesser extent via HvHKT2;1 and AtAKT1, was inhibited by increasing concentrations of NH4+. Together, the results of this study show that plant K+ transporters and channels are able to transport NH4+. Unregulated NH4+ uptake via these transporters may contribute to NH4+ toxicity at low K+ levels, and may explain the alleviation of NH4+ toxicity by K+.