To elucidate the role of NH4+ transporters in N nutrition of tomato, two new NH4+ transporter genes were isolated from cDNA libraries of root hairs or leaves of tomato. While LeAMT1;2 is closely related to LeAMT1;1 (75.6% amino acid identity), LeAMT1;3 is more distantly related (62.8% identity) and possesses two short upstream open reading frames in the 5' end of the mRNA and a particularly short N-terminus of the protein as unique features. When expressed in yeast mutants defective in NH4+ uptake, all three genes complemented NH4+ uptake. In roots of hydroponically grown plants, transcript levels of LeAMT1;2 increased after NH4+ or NO3- supply, while LeAMT1;1 was induced by N deficiency coinciding with low glutamine concentrations, and LeAMT1;3 was not detected. In aeroponic culture, expression of LeAMT1;1 and LeAMT1;2 was higher in root hairs than in the remaining root fraction. Growth of plants at elevated CO2 slightly decreased expression of LeAMT1;2 and LeAMT1;3 in leaves, but strongly repressed transcript levels of chloroplast glutamine synthetase and photorespiratory serine hydroxymethyl-transferase. Expression of LeAMT1;2 and LeAMT1;3 showed a reciprocal diurnal regulation with highest transcript levels of LeAMT1;3 in darkness and highest levels of LeAMT1;2 after onset of light. These results indicate that in tomato at least two high-affinity NH4+ transporters, LeAMT1;1 and LeAMT1;2, are differentially regulated by N and contribute to root hair-mediated NH4+ acquisition from the rhizosphere. In leaves, the reciprocally expressed transporters LeAMT1;2 and LeAMT1;3 are supposed to play different roles in N metabolism, NH4+ uptake and/or NH3 retrieval during photorespiration.