Ammonium (NH(4)(+)) toxicity is a significant agricultural problem globally, compromising crop growth and productivity in many areas. However, the molecular mechanisms of NH(4)(+) toxicity are still poorly understood, in part due to a lack of valuable genetic resources. Here, a novel Arabidopsis mutant, amos2 (ammonium overly sensitive 2), displaying hypersensitivity to NH(4) (+) in both shoots and roots, was isolated. The mutant exhibits the hallmarks of NH(4)(+) toxicity at significantly elevated levels: severely suppressed shoot biomass, increased leaf chlorosis, and inhibition of lateral root formation. Amos2 hypersensitivity is associated with excessive NH(4)(+) accumulation in shoots and a reduction in tissue potassium (K(+)), calcium (Ca(2+)), and magnesium (Mg(2+)). We show that the lesion is specific to the NH(4)(+) ion, is independent of NH(4)(+) metabolism, and can be partially rescued by elevated external K(+). The amos2 lesion was mapped to a 16-cM interval on top of chromosome 1, where no similar mutation has been previously mapped. Our study identifies a novel locus controlling cation homeostasis under NH(4)(+) stress and provides a tool for the future identification of critical genes involved in the development of NH(4)(+) toxicity.