Screening was performed to isolate microorganisms which accumulate trehalose. Of 96 yeast strains screened, a basidiomycotinous yeast, Filobasidium floriforme IFO 1916, accumulated the largest amount of trehalose. Under optimum conditions in a 5-l jar fermenter, F. floriforme IFO 1916 accumulated 10 mg/ml trehalose (dry cell weight, 52 mg/ml; trehalose content, 20.0%) from 100 mg/ml glucose after cultivation for 24 h. Strain T12, a morphological and trehalose non-assimilating mutant of F. floriforme IFO 1916, accumulated 30% more trehalose than the parent strain.