The antitumor β-glucan from the fruit body of Grifola frondosa was resolved by DEAE-Sephadex chromatography into neutral and acidic components, both of which were found to be growth-inhibitory against sarcoma 180 solid tumor inplanted in ICR male mice (Ohno et al., Chem. Pharm. Bull., 33, 1181 (1984)). In the present study, the acidic β-glucan was further fractionated by diethylaminoethyl (DEAE)-Sephadex chromatography followed by ammonium sulfate precipitation, and the resultant β-glucan subfractions were assayed for antitumor effect.The β-glucan fraction absorbed on DEAE-Sephadex A-25 was eluted with 8 M urea, an inducer of helix-random coil transition of β-1, 3-glucan. The eluted glucan fraction (fraction N (8M)) was a β-1, 3-glucan branched at the 6 position and possessing almost the same properties as those of a neutral β-1, 3-glucan obtained from the neutral fraction (fraction N (0M)). The remaining acidic β-glucan fraction was eluted with 0.45 M ammonium bicarbonate in 8 M urea (fraction A), and was further separated into a β-1, 3-glucan branched at the 6 position (fraction AP) and a β-1, 6-glucan (fracton AS) fractions by ammonium sulfate precipitation. Fraction AP showed potent antitumor activity, while that of fraction AS was quite weak. These results suggest that G. frondosa contains three kinds of acidic glucans, (1) branched β-1, 3-glucan non covalently coupled with an acidic component, (2) branched β-1, 3-glucan tightly coupled with an acidic component, and (3) acidic β-1, 6-glucan, and that the antitumor activity of the crude extracts of G. frondosa is due to both the neutral and the acidic β-1, 3-glucans.