Rhizospheric nitric oxide (NO) and carbon dioxide (CO2) are signalling compounds known to affect physiological processes in plants. Their joint influence on tree nitrogen (N) nutrition, however, is still unknown. Therefore, this study investigated, for the first time, the combined effect of rhizospheric NO and CO2 levels on N uptake and N pools in European beech (Fagus sylvatica L.) seedlings depending on N availability. For this purpose, roots of seedlings were exposed to one of the nine combinations (i.e., low, ambient, high NO plus CO2 concentration) at either low or high N availability. Our results indicate a significant effect of rhizospheric NO and/or CO2 concentration on organic and inorganic N uptake. However, this effect depends strongly on NO and CO2 concentration, N availability and N source. Similarly, allocation of N to different N pools in the fine roots of beech seedlings also shifted with varying rhizospheric gas concentrations and N availability.