Amino acid transport in plants is mediated by at least two large families of plasma membrane transporters. Arabidopsis thaliana, a nonmycorrhizal species, is able to grow on media containing amino acids as the sole nitrogen source. Arabidopsis amino acid permease (AAP) subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in long-distance transport between organs. We show that the broad-specificity, high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER1 (LHT1), an AAP homolog, is expressed in both the rhizodermis and mesophyll of Arabidopsis. Seedlings deficient in LHT1 cannot use Glu or Asp as sole nitrogen sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue. These findings are consistent with two major LHT1 functions: uptake in roots and supply of leaf mesophyll with xylem-derived amino acids. The capacity for amino acid uptake, and thus nitrogen use efficiency under limited inorganic N supply, is increased severalfold by LHT1 overexpression. These results suggest that LHT1 overexpression may improve the N efficiency of plant growth under limiting nitrogen, and the mutant analyses may enhance our understanding of N cycling in plants.