On Zn-polluted soils, populations of the ectomycorrhizal basidiomycete Suillus bovinus exhibit an elevated Zn tolerance when compared to populations on non-polluted sites. To elucidate the mechanism of Zn tolerance, the time-course of Zn uptake was studied in isolates with contrasting Zn tolerance. Unidirectional fluxes and subcellular compartmentation of Zn were investigated through radiotracer flux analyses. Fluorescence imaging was used to support the subcellular Zn compartmentation. After 2 h of exposure to 200 μM Zn, significantly more Zn was accumulated in Zn-sensitive isolates compared to tolerant isolates, despite similar short-term uptake kinetics and similar extracellular Zn sequestration in cell walls. In Zn-sensitive isolates twice as much Zn accumulated in the cytoplasm and 12 times more Zn in the vacuole. (65)Zn efflux analyses revealed a considerably faster Zn export in the Zn-tolerant isolate. The adaptive Zn tolerance in S. bovinus is therefore achieved by a preferential removal of Zn out of the cytoplasm, back into the apoplast, instead of the usual transfer of Zn into the vacuole. Zn exclusion in the fungal symbiont eventually contributes to a lower Zn influx in host plants.