Fungal colonization can significantly affect the secondary metabolism of the host plants. We tested the impact of a common below-ground symbiosis, i.e., ectomycorrhiza formation, on poplar leaf chemical components that are involved in the defence against a common disease, i.e., rust fungi, in N-deficient soil. A rust-susceptible poplar clone (Populus trichocarpa × deltoides 'Beaupré') was (a) non-associated with ectomycorrhizal fungus (EM) Hebeloma mesophaeum (Pers.) Quélet MÜN and non-infected with rust fungus Melampsora larici-populina Kleb. (isolate 98AG31), (b) associated with EM, (c) inoculated with rust fungus and (d) associated with EM and inoculated with rust fungus. Poplar leaves were analysed by photometric and mass spectrometric techniques (liquid chromatography-tandem mass spectrometry (LC-MS/MS), pyrolysis-field ionization mass spectrometry (Py-FIMS)). Both rust infection and mycorrhiza formation led to increased proportions of condensed tannins in relation to total phenolics (13% in the control, 18-19% in the fungal treatments). In contrast, salicylic acid concentration (6.8 µg g(-1) in the control) was higher only in the rust treatments (17.9 and 25.4 µg g(-1) with rust infection). The Py-FIMS analysis revealed that the rust-infected treatments were significantly separated from the non-rust-infected treatments on the basis of six flavonoids and one lipid. The relative abundance of these components, which have known functions in plant defence, was decreased after rust infection of non-mycorrhizal plants, but not in mycorrhizal plants. The results indicate that the ectomycorrhizal formation compensated the rust infection by a decrease in the flavonoid syntheses. The study provides new evidence for an interactive response of mycorrhizal colonization and infection with rust fungi in the metabolism of poplar.