Metal tolerance of filamentous fungi is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the ectomycorrhizal fungal model Hebeloma cylindrosporum, we carried out a functional screening of an H. cylindrosporum cDNA library in the zrc1D mutant strain of Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. This strategy allowed the isolation of HcZnT1, a gene belonging to the cation diffusion facilitator family, which induced tolerance to Zn, but not to other metals. HcZnT1 was constitutively expressed in Hebeloma cells, whatever the Zn status of the medium and the fungal cell type (mycelia, sporocarps, mycorrhizas). A HcZnT1:GFP fusion protein was expressed in yeast and the corresponding fluorescence was recorded on endoplasmic reticulum membranes. Taken together, these different findings suggest a dual role of HcZnT1 in Zn homeostasis of fungal cells, by supplying requested Zn ions for the functioning of the endoplasmic reticulum as well as by detoxifying the cytosol under Zn stress. Zn pools were also investigated by using the Zn-specific fluoro-phore zinquin in H. cylindrosporum cells. Zinquin labeling revealed compartmentalization in intracellular vesicles interspersed throughout the cytoplasm that do not correspond to vacuolar compartments. Altogether the present data represent the first steps into the understanding of Zn homeostasis and tolerance in Hebeloma.