Agrocybe cylindracea, an edible mushroom belonging to Bolbitiaceae, Agaricales, is widely used as invaluable medicinal material in the oriental countries. This study was initiated to find the genes expressed during the fruiting body formation of A. cylindracea. The cDNAs expressed differentially during fruiting body morphogenesis of A. cylindracea were isolated through subtractive hybridization between vegetative mycelia and fruiting bodies. The cDNAs expressed in the fruiting body morphogenesis of A. cylindracea were cloned and twenty genes were identified. Eleven were homologous to genes of known functions, three were homologous to genes in other organism without any function known. Six were completely novel genes specific to A. cylindracea so far examined. Some genes with known functions were a pleurotolysin, a self-assembling poreforming cytolysins; Aa-Pril and Pir2p, specifically induced genes during fruiting initiation of other mushroom, Agrocybe aegerita; an amino acid permease; a cytochrome P450; a MADS-box gene; a peptidylprolyl isomerase; and a serine proteinase. For other clones, no clear function was annotated so far. We believe the first report of the differentially expressed genes in fruiting process of A. cylindracea will be great helps for further research.