Hydrophobins are fungal cell wall proteins which play a crucial role in cell adhesion and aggregative processes. We have identi®ed a new hydrophobin cDNA (hydPt-3) in the symbiotic mycelium of Pisolithus tinctorius (putative P. albus) during the formation of ectomycorrhizae around eucalypt roots. This sequence is highly divergent from two other previously identi®ed Pisolithus symbiosis-regulated hydrophobins, hydPt-1 and hydPt-2. Also, expression analyses demonstrated that hydPt-3 is up-regulated during the formation of ectomycorrhizae. In contrast to phytopathogenic fungi, changes in glucose or ammonium concentrations in the growth medium did not in¯uence the accumulation of any Pisolithus hydrophobin mRNAs. This suggests that other factors act as regulators of hydrophobin gene expression in ectomycorrhizae.