Differential screening of a cDNA library was used to clone genes that are specifically expressed during mushroom development in the basidiomycete Agaricus bisporus. One of the isolated genes encodes a polypeptide of 112 amino acid residues and belongs to the fungal gene family encoding hydrophobins. This gene, hypA, has the characteristic pattern of eight cysteine residues at conserved positions and a hydrophobicity pattern that is very similar to class I hydrophobins. Elucidation of the genomic structure of hypA led to the identification of a second copy, hypC, located downstream of hypA. Although at a much lower level, hypC is like hypA specifically expressed on fruit bodies. The hypA mRNA level is transiently increased ten days after fruit body induction and expression appears to be associated with rapid expansion of the mushroom caps. In mushroom caps, very high concentrations of hypA messengers were found in the (outer) peel tissue, where they accumulate to more than 60% of the total mRNA mass. The corresponding protein with a molecular mass of 8 to 9 kDa was purified from this peel tissue and was identified by N-terminal sequencing. Our results suggest that HYPA forms a protective hydrophobic layer instrumental in cap formation.