The formation of the ectomycorrhiza implies an alteration in gene expression of both the plant and fungal partners, a process which starts before the formation of any symbiotic interface. However, little is known on the regulation pattern occurring in different parts of the root system. Our experimental system consisting of a micropropagated oak with a hierarchical root system was shown to exhibit symbiosis functional traits prior to any mycorrhizal tissue differentiation after the inoculation with the basidiomycete Piloderma croceum. Using a cDNA array, the plant gene regulation was analyzed in the pre-mycorrhizal phase. Seventy-five transcripts showed differential expression in pre-mycorrhizal lateral and principal roots, and both root types exhibited different sets of responsive genes. For transcripts selected according to a statistical analysis, the alteration in gene expression was confirmed by RT-PCR and quantitative real-time PCR. Genes regulated in pre-mycorrhizal lateral roots displayed an almost identical expression in mycorrhizas. In contrast, genes regulated in pre-mycorrhizal principal roots were often regulated differently in ectomycorrhizas. Down-regulation affected most of the regulated genes involved in metabolism, whereas most of the regulated genes related to cell rescue functions, water regulation and defence response were up-regulated. Regulation of such genes could explain the increase of global resistance observed in mycorrhizal plants.