The hfb1 gene of the filamentous fungus Trichoderma reesei, previously cloned as a gene which was abundantly expressed when the fungus was grown on glucose-containing medium, was shown to encode a novel fungal hydrophobin. The encoded 97-amino-acid protein is cysteine-rich and has a typical signal sequence for secretion. Signal-sequence cleavage and putative proteolytic processing results in the mature HFBI protein of 75 amino acids. Antibodies raised against the HFBI protein expressed in Escherichia coli detected the T. reesei HFBI protein in the fungal cell wall and in the culture medium of submerged glucose-containing cultures. The identity of HFBI was verified by N-terminal and peptide sequencing of proteins purified both from the cell wall and culture medium. In the cell wall most of the HFBI formed SDS-insoluble complexes that could be extracted with trifluoroacetic acid. Bubbling or freezing of the culture medium caused HFBI to form aggregates that coprecipitated with a yellow pigment produced by the fungus.