Three different hydrophobins (Vmh1, Vmh2, and Vmh3) were isolated from monokaryotic and dikaryotic vegetative cultures of the edible fungus Pleurotus ostreatus. Their corresponding genes have a number of introns different from those of other P. ostreatus hydrophobins previously described. Two genes (vmh1 and vmh2) were expressed only at the vegetative stage, whereas vmh3 expression was also found in the fruit bodies. Furthermore, the expression of the three hydrophobins varied significantly with culture time and nutritional conditions. The three genes were mapped in the genomic linkage map of P. ostreatus, and evidence is presented for the allelic nature of vmh2 and POH3 and for the different locations of the genes coding for the glycosylated hydrophobins Vmh3 and POH2. The glycosylated nature of Vmh3 and its expression during vegetative growth and in fruit bodies suggest that it should play a role in development similar to that proposed for SC3 in Schizophyllum commune. Many hydrophobins have been described since SC1 and SC3 were found (16,44), and their peculiar characteristics have beendescribed (15,54). In the pathogenic fungus Cladosporium fulvum, six hydrophobin genes encode both class I (53) and class II (51) hydrophobins, and this seems to be the highest hydrophobin gene number in an individual fungus. At least four hydrophobin genes have been found in Schizophyllum commune, playing different roles during development (47): SC3 is expressed in monokaryons and dikaryons while the others are active only in dikaryons. For the edible mushroom Agaricus bisporus, various genes have also been identified coding for hydrophobins, ABH1/HypA and ABH2 in fruit bodies (12, 27) and ABH3 in vegetative mycelia (28), which probably perform different functions. Hydrophobin gene redundancy opens the question about their number and different physiological functions.Gene disruption was used to verify the roles played by SC3 and SC4 in the formation of dikaryon aerial structures (47). The regulation of hydrophobin gene expression has been found to occur under the control of a complex set of factors, such as mating type in S. commune SC1 and SC3 (51) or nutrient availability and starvation in Neurospora crassa gene eas (9), Magnaporthe grisea MPG1 (26, 46), Trichoderma reesei hf1 and hfb2 (33), and C. fulvum HCF-1 to HCF-6 (43).Pleurotus ostreatus (Jacq. ex Fr) Kummer is a commercially important edible mushroom commonly known as the oyster mushroom. This fungus is industrially produced as human food, and it accounts for nearly a quarter of the world mushroom production (10). It is also used for the bioconversion of agricultural, industrial, and lignocellulose wastes (7, 37) as a source of enzymes and other chemicals for industrial and medical applications (18,19,30), as an agent for bioremediation (6), and as organic fertilizer (1). Several different P. ostreatus varieties are industrially produced. Commercial varieties florida and ostreatus differ in size, color, and temperature tolerance. Both varieties have been used in prev...