Despite the important role of the ammonium ion in metabolism, i.e. as a form of nitrogen that is taken up from the soil by microorganisms and plants, little is known at the molecular level about its transport across biomembranes. Biphasic uptake kinetics have been observed in roots of several plant species. To study such transport processes, a mutant yeast strain that is deficient in two NH4+ uptake systems was used to identify a plant NH4+ transporter. Expression of an Arabidopsis cDNA in the yeast mutant complemented the uptake deficiency. The cDNA AMT1 contains an open reading frame of 501 amino acids and encodes a highly hydrophobic protein with 9-12 putative membrane spanning regions. Direct uptake measurements show that mutant yeast cells expressing the protein are able to take up [14C]methylamine. Methylamine uptake can be efficiently competed by NH4+ but not by K+. The methylamine uptake is optimal at pH 7 with a Km of 65 microM and a Ki for NH4+ of approximately 10 microM, is energy-dependent and can be inhibited by protonophores. The plant protein is highly related to an NH4+ transporter from yeast (Marini et al., accompanying manuscript). Sequence homologies to genes of bacterial and animal origin indicate that this type of transporter is conserved over a broad range of organisms. Taken together, the data provide strong evidence that a gene for the plant high affinity NH4+ uptake has been identified.