Although the function of the extramatrical mycelium of ectomycorrhizal fungi is considered essential for the acquisition of nitrogen by forest trees, gene regulation in this fungal compartment is poorly characterized. In this study, the expression of the nitrate transporter gene nrt2 from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was shown to be regulated by plant host and carbon sources. In the presence of a low fructose concentration, nrt2 expression could not be detected in the free-living mycelium but was high in the extramatrical symbiotic mycelium associated to the host plant Pinus pinaster. In the absence of nitrogen or in the presence of nitrate, high sugar concentrations in the medium were able to enhance nrt2 expression. Nevertheless, in the presence of high fructose concentration, high ammonium concentration still completely repressed nrt2 expression indicating that the nitrogen repression overrides sugar stimulation. This is the first report revealing an effect of host plant and of carbon sources on the expression of a fungal nitrate transporter-encoding gene.