The hfb2 gene encoding the hydrophobin HFBII of the filamentous fungus Trichoderma reesei was isolated by heterologous hybridization using the vegetative hydrophobin I, hfb1, gene of T. reesei as a probe. The hfb2 gene codes for a typical fungal secreted hydrophobin of 71 amino acids containing eight cysteine residues. The amino acid similarity towards HFBI is 69%. The HFBII protein was isolated from the fungal spores by extraction with trifluoroacetic acid/acetonitrile solution, and by bubbling from the lactose‐based culture medium. Expression of the hfb1 and hfb2 genes is divergent, hfb1 expression was only observed in vegetative cultures on glucose‐containing and sorbitol‐containing media. It was not expressed on media containing complex plant polysaccharides, cellulose, xylan, cellobiose or lactose, whereas hfb2 was highly expressed in vegetative cultures on these media. Expression of hfb2 was also strongly induced by N and C starvation, by light and in conidiating cultures.