Many characteristics of fungal hydrophobins, such as an ability to change hydrophobicity of different surfaces. have potential for several applications. The large-scale processes of production and isolation of these proteins susceptible to aggregation and attachment to interfacial surfaces still needs to be studied. We report for the first time on a method for a gram-scale production and purification of a hydrophobin, HFBI of Trichoderma reesei. A high production level of the class II hydrophobin (0.6 g 1(-1)) was obtained by constructing a T reesei HFBI-overproducing strain containing three copies of the hfb1 gene. The strain was cultivated on glucose-containing medium. which induces expression of hfb1. HFBI hydrophobin was purified from the cell walls of the fungus because most of the HFBI was cell-bound (80%). Purification was carried out with a simple three-step method involving extraction of the mycelium with 1% SDS at pH 9.0, followed by KCI precipitation to remove SDS, and hydrophobic interaction chromatography. The yield was 1.8 g HFBI from mycelium (419 g dw), derived from 151 of culture. HFBI was shown to be rather unstable to N-terminal asparagine deamidation and also., to some extent, to non-specific proteases although its thermostability was excellent.