Hydrophobins are fungal cell wall proteins which play a crucial role in cell adhesion and aggregative processes. We have identified 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 identified 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 influence the accumulation of any Pisolithus hydrophobin mRNAs. This suggests that other factors act as regulators of hydrophobin gene expression in ectomycorrhizae.