AtAMT2 is an ammonium transporter that is only distantly related to the five members of the AtAMT1 family of high-affinity ammonium transporters in Arabidopsis. The short-lived radioactive ion (13)NH(4)(+) was used to show that AtAMT2, expressed in yeast (Saccharomyces cerevisiae), is a high-affinity transporter with a K(m) for ammonium of about 20 microM. Changes in external pH between 5.0 and 7.5 had little effect on the K(m) for ammonium, indicating that NH(4)(+), not NH(3), is the substrate for AtAMT2. The AtAMT2 gene was expressed in all organs of Arabidopsis and was subject to nitrogen (N) regulation, at least in roots where expression was partially repressed by high concentrations of ammonium nitrate and derepressed in the absence of external N. Although expression of AtAMT2 in shoots responded little to changes in root N status, transcript levels in leaves declined under high CO(2) conditions. Transient expression of an AtAMT2-green fluorescent protein fusion protein in Arabidopsis leaf epidermal cells indicated a plasma membrane location for the AtAMT2 protein. Thus, AtAMT2 is likely to play a significant role in moving ammonium between the apoplast and symplast of cells throughout the plant. However, a dramatic reduction in the level of AtAMT2 transcript brought about by dsRNA interference with gene expression had no obvious effect on plant growth or development, under the conditions tested.