Ammonium and nitrate are the prevalent nitrogen sources for growth and development of higher plants. 15N-uptake studies demonstrated that ammonium is preferred up to 20-fold over nitrate by Arabidopsis plants. To study the regulation and complex kinetics of ammonium uptake, we isolated two new ammonium transporter (AMT) genes and showed that they functionally complemented an ammonium uptake-deficient yeast mutant. Uptake studies with 14C-methylammonium and inhibition by ammonium yielded distinct substrate affinities between </=0.5 and 40 microM. Correlation of gene expression with 15NH4+ uptake into plant roots showed that nitrogen supply and time of day differentially regulated the individual carriers. Transcript levels of AtAMT1;1, which possesses an affinity in the nanomolar range, steeply increased with ammonium uptake in roots when nitrogen nutrition became limiting, whereas those of AtAMT1;3 increased slightly, with AtAMT1;2 being more constitutively expressed. All three ammonium transporters showed diurnal variation in expression, but AtAMT1;3 transcript levels peaked with ammonium uptake at the end of the light period, suggesting that AtAMT1;3 provides a link between nitrogen assimilation and carbon provision in roots. Our results show that high-affinity ammonium uptake in roots is regulated in relation to the physiological status of the plant at the transcriptional level and by substrate affinities of individual members of the AMT1 gene family.