Nuclear magnetic resonance spectroscopy was used to monitor 15NH4+ assimilation and amino acid biosynthesis in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton. (strain S238). In mycelium growing rapidly on 15NH4+, [amido-15N]glutamine was the major 15N-labelled species. When 15N-labelled mycelium was transferred into medium containing 14NH4+, the resonance for [amino-15N]glutamine decreased with a half-life of about 3.0 h, whereas the resonance for [amino-15N]glutamine remained unchanged. Such behaviour is consistent with glutamine synthetase (GS) being the major route of 15NH4+ assimilation. However, the higher accumulation of [15N]alanine observed in the presence of the GS inhibitor, methionine sulfoximine, indicated that a part of the glutamate pool was formed by the glutamate dehydrogenase (GDH) pathway. When the mycelium was in stationary phase (i.e. low extracellular NH4+), the intramolecular 15N labelling of glutamine suggested that the GDH and GS pathways were simultaneously assimilating NH4+. The N supply and the growth stage, therefore, influence the expression of the activities of GDH and GS. The current isotopic data identify other fates of absorbed 15N: glutamate decarboxylation gives rise to γ-aminobutyrate; transamination between glutamate and pyruvate yields alanine; and arginine accumulates. It is concluded that GS is the main pathway of primary assimilation of NH4+ in L. bicolor, but GDH may also contribute significantly to this process.