In temperate forests, the roots of various tree species are colonized by ectomycorrhizal fungi, which have a key role in the nitrogen nutrition of their hosts. However, not much is known about the molecular mechanisms related to nitrogen metabolism in ectomycorrhizal plants. This study aimed to evaluate the nitrogen metabolic response of oak plants when inoculated with the ectomycorrhizal fungus Pisolithus tinctorius. The expression of candidate genes encoding proteins involved in nitrogen uptake and assimilation was investigated in ectomycorrhizal roots. We found that three oak ammonium transporters were over-expressed in root tissues after inoculation, while the expression of amino acid transporters was not modified, suggesting that inorganic nitrogen is the main form of nitrogen transferred by the symbiotic fungus into the roots of the host plant. Analysis by heterologous complementation of a yeast mutant defective in ammonium uptake and GFP subcellular protein localization clearly confirmed that two of these genes encode functional ammonium transporters. Structural similarities between the proteins encoded by these ectomycorrhizal upregulated ammonium transporters, and a well-characterized ammonium transporter from E. coli, suggest a similar transport mechanism, involving deprotonation of NH₄⁺, followed by diffusion of uncharged NH₃ into the cytosol. This view is supported by the lack of induction of NH₄⁺ detoxifying mechanisms, such as the GS/GOGAT pathway, in the oak mycorrhizal roots.