The development of ectomycorrhizal symbiosis leads to drastic changes in gene expression in both partners. However, little is known about the spatial regulation of symbiosis-regulated genes. Using cDNA array profiling, we compared the levels of expression of fungal genes corresponding to approximately 1,200 expressed sequenced tags in the ectomycorrhizal root tips (ECM) and the connected extraradical mycelium (EM) for the Paxillus involutus-Betuld pendula ectomycorrhizal association grown on peat in a microcosm system. Sixty-five unique genes were found to be differentially expressed in these two fungal compartments. In ECM, a gene coding for a putative phosphatidylserine decarboxylase (Psd) was up-regulated by 24-fold, while genes coding for urea (Dur3) and spermine (Tpo3) transporters were up-regulated 4.1- and 6.2-fold in EM. Moreover, urea was the major nitrogen compound found in EM by gas chromatography-mass spectrometry analysis. These results suggest that (i) there is a spatial difference in the patterns of fungal gene expression between ECM and EM, (ii) urea and polyamine transporters could facilitate the translocation of nitrogen compounds within the EM network, and (iii) fungal Psd may contribute to membrane remodeling during ectomycorrhiza formation.