Agaricus blazei is a medically important mushroom widely eaten and prescribed in Japan. Polysaccharide fractions were prepared from cultured A. blazei by repeated extraction with hot water (AgHWE), cold NaOH (AgCA), and then hot NaOH (AgHA). By chemical, enzymic, and NMR analyses, the primary structures of AgHWE, AgCA, and AgHA were mainly composed of 1,6-beta-glucan. Among these fractions, the NaOH extracts showed antitumor activity against the solid form of Sarcoma 180 in ICR mice. To demonstrate the active component in these fractions, several chemical and enzymic treatments were applied. These fractions were found to be i) neutral beta-glucan passing DEAE-Sephadex A-25, ii) resistant to periodate oxidation (I/B) and subsequent partial acid hydrolysis (I/B/H), iii) resistant to a 1,3-beta-glucanase, zymolyase, before I/B, but sensitive after I/B/H. In addition, after I/B/H treatment of the neutral fraction of AgCAE, a signal around 86 ppm attributable to 1,3-beta glucosidic linkage was detectable in the 13C-NMR spectrum. These facts strongly suggest that a highly branched 1,3-beta-glucan segment forms the active center of the antitumor activity.