Mushroom development is important for both basic scientific research and for practical mushroom production. In previous screening studies of natural compounds that are effective for fruiting body development of oyster mushrooms (Pleurotus ostreatus) on agar medium, we identified a triterpenoid saponin that exerted a hormone-like effect. Not only did the saponin stimulate mushroom development, but P. ostreatus also changed its morphology as the concentration of saponin in the medium increased. We have synthesized a betulin glycoside by linking a sugar moiety to a triterpenoid betulin derived from outer bark of Betulla platyphylla. The sugar chain was further lengthened by transglycosylation using cyclodextrin glycosyltransferase (CGTase) of Bacillus macerans. Neither betulin nor the triterpenoid itself stimulated fruiting body development, but the activity was generated as a sugar moiety was introduced onto betulin. Fruiting activity correlated with the sugar length, indicating that the presence of the sugar moiety was indispensable for fruiting activity. We proposed that an as yet unknown hydrophobic compound with an attached sugar chain triggers fruiting in P. ostreatus. We synthesized a 3-O-alkyl-D-glucose (AG) and determined that AG with an eight-carbon alkyl chain effectively induced fruiting bodies for P. ostreatus. Thus, a sugar-containing amphipathic compound can stimulate fruiting body development. © 2009 Bentham Science Publishers Ltd.