The basidiomycetous mushroom Lentinula edodes (Shiitake) exhibits a unique process of cell differentiation termed "fruiting-body formation". To clarify the relationship between membrane lipids and fruiting-body formation in this fungus, we investigated variations in levels of phospholipids, cerebrosides, fatty acyl residues in the major phospholipids, and fatty acyl and sphingoid base residues in cerebrosides during vegetative growth and fruiting-body formation. PC, PE, and PS were the primary phospholipids in the cells of L. edodes. After a shift in growth temperature of L. edodes mycelia has been shifted from 25 to 18 degrees C, the proportion of unsaturated FA (UFA), such as linoleic acid (18:2) and oleic acid (18:1), increased. In contrast, during fruiting-body formation induced by the temperature downshift to 18 degrees C, 18:2 of PC in the primordia and fruiting bodies decreased, and the UFA of PE and 18:1 of PC increased compared with the proportions in mycelia growing at 18 degrees C. These results showed that the proportions of fatty acyl residues in PC and PE differed during fruiting-body formation in L. edodes. Moreover, the amount of cerebrosides in primordia increased compared with those in mycelia and fruiting bodies and, in these differentiating tissues, the proportion of 2-hydroxypentadecanoic acid increased whereas that of 2-hydroxyoctadecanoic acid decreased compared with that in the mycelia. However, the proportion of sphingoid base residues in cerebrosides did not change during fruiting-body formation in L. edodes.