To investigate the impact of organic N compounds for inorganic nitrogen uptake in the rhizosphere, we fed ammonium nitrate with or without amino acids (i.e., glutamine or arginine) to the roots of non-mycorrhizal beech (Fagus sylvatica L.) seedlings under controlled conditions at different levels of N availability. Uptake of individual N sources was determined from ¹⁵N (inorganic N) and ¹⁵N ¹³C (organic N) accumulation in the roots. In addition, gene fragments encoding proteins involved in N uptake and metabolism were cloned from beech for gene expression analyses by quantitative real-time PCR in the roots. Generally, ammonium was preferred over nitrate as N source. Organic N sources were taken up by beech roots as intact molecules. Uptake of organic N was significantly higher than inorganic N uptake, thus contributing significantly to N nutrition of beech. Depending on the level of N availability, inorganic N uptake was negatively affected by the presence of organic N sources. This result indicates an overestimation of the contribution of inorganic N uptake to N nutrition of beech in previous studies. Apparently, association with mycorrhizal fungi is not essential for organic N uptake by beech roots. Gene expression analyses showed that transcriptional regulation of the amino acid transporters FsCAT3, FsCAT5, FsAAT and FsAAP and the ammonium transporter FsAMT1.2 in the roots is involved in N nutrition of beech.