The initiation process of effector mechanism of the antitumor activity of (1→3)-β-D-glucans has been examined in detail by applying treatment of mice with (1→3)-β-D-glucanases, kitalase (KIT) and zymolyase (ZYM), via i.p. route after i.p. administration of (1→3)-β-D-glucans, grifolan (GRN) and CM-curdlan (CM-CUD). A significant decrease of antitumor activity caused by GRN was observed by treatment with KIT within 48 h, and that caused by GRN administered via i.v. route was also reduced by the i.v. injection of KIT, although it was less effective than i.p. treatment. Pretreatment of mice with thioglycollate reduced the period of time to abrogate antitumor activity by the sequential treatment of KIT. Similar results were also observed on ZYM treatment after i.p. treatment of CM-CUD. These facts suggest that the period of manifestation of the antitumor activity by (1→3)-β-D-glucans must be longer than a few days.