Grifola frondosa is an edible mushroom, which has a high value in health research. Hydrophobins are small proteins with remarkable surface-active properties, which make hyphal wall surfaces water-repellent. The aim of this study was to identify and characteristic more hydrophobin genes from the hypha of G. frondosa. In this study, a cDNA library of the hyphae was constructed. Two new hydrophobin genes, which were named hgfIII and hgfIV, respectively, were isolated and characterized. The gene cDNA sequences, deduced amino acid sequences, primary structures and hydropathy plots of the hydrophobin proteins were analyzed. Results showed that the deduced protein amino acid sequences of two genes shared the same hydrophobicity patterns as HGFI, a class I hydrophobin already known from G. frondosa. The construction of a phylogenetic tree indicated that the two newly-discovered hydrophobins formed separate clades with HGFI. The stage of culture was found to have a marked influence on gene expression between hgfI and both hgfIII and hgfIV. Transcription patterns demonstrated that the expression of the novel hgfIII was highly induced in hypha compared with that of hgfI

and hgfIV. However, hgfIV was highly induced in fruiting bodies among these genes. The diverse transcription patterns of the hydrophobin genes suggested that they may play different roles in different growth stages of G.frondosa.