The majority of cellobiase activity in Termitomyces clypeatus was localized in vacuolar fractions of the fungus under secretory and nonsecretory conditions of growth. Activities of marker proteins for subcellular organelles, e.g., vacuoles, cytosol, ER, and mitochondria, in mycelial extracts from the secreting conditions increased by approximately 20, 12, 5, and 2.5 times, respectively, as compared to those obtained from mycelium. grown in nonsecreting conditions. The average size and concentration of vacuoles visualized by electron microscopy were also increased in secreting conditions in the fungus. The specific activity of cellobiase in vacuoles isolated in Ficoll-sucrose gradient, as obtained from mycelial growth in secretory medium, was more than 40 times higher in comparison to that found from nonsecretory medium. The results indicated that subcellular localization of cellobiase in vacuoles is regulated by the cellular signaling prevailing in the fungus. Mycelial extraction of intracellular proteins by hand grinding and by bead-beater from cells frozen in the presence or absence of liquid nitrogen was also compared. Maximum recovery of intracellular protein was obtained with the bead-beater under aerobic conditions in the absence of nitrogen. Highest recovery of vacuoles up to 85% was obtained by single-step ultracentrifugation of the mycelial extract of the fungus in Ficoll-sucrose gradient. The method appeared to be useful for separation of other subcellular organelles in filamentous fungi.