Augmentation of antitumor activity by combined in situ freeze-destruction of tumor (cryosurgery) and administration of antitumor active substances isolated from a hot water extract of an edible mushroom, Flammulina velutipes (Curt. ex Fr.) SING., was investigated in sarcoma 180 bearing ICR mice. Antitumor active substances of the mushroom included beta-(1,3)-glucan (EA3) and protein-bound polysaccharide (EA6). Antitumor activity was evaluated by the growth rate of the solid tumor rechallenged subcutaneously (s.c.) or by cumulative mortalities of the mice rechallenged intraperitoneally (i.p.) with the ascites tumor, on day 7 after cryosurgery. Weak antitumor effect induced by cryosurgery against challenged solid tumor of sarcoma 180 was markedly augmented by i.p. administration of EA6 (10 mg/kg). Cryosurgery of the solid sarcoma 180 apparently did not induce any antitumor effect against challenged ascites sarcoma 180. However, when cryosurgery was combined with oral administration of the polysaccharides (50 mg/kg), prolonged survival of the mice challenged with ascites sarcoma 180 i.p. was recognized by EA6, but not by EA3. Timing of the administration of EA6 (i.p.) with cryosurgery was best in pre- and post-cryosurgery. Immunological activity of EA6 to the host was discussed.