A 21-kDa heteropolysaccharide, coded as GFPS1b, was obtained from the cultured mycelia of Grifola frondosa GF9801 by hot-water extraction, ethanol precipitation, and fractioned by DEAE Sepharose Fast-flow, followed by the purification with Sephadex G-100 column chromatography using an AKTA purifier. It exhibited more potent anti-proliferative activity on MCF-7 cells than other polysaccharide fractions. GFPS1b was an acidic polysaccharide with approximately 16.60% protein and 4.3% uronic acid. Gas chromatography of absolute acid hydrolysate of GFPS1b suggested that it was composed Of D-glucose, D-galactose, and L-arabinose with a molar ratio of 4:2:1. Periodate oxidation, Smith degradation, partial acid hydrolyzation, methylation analysis, FT-IR, and H-1, C-13 NMR spectroscopy analysis revealed that GFPS1b had a backbone consisting of alpha-(1 -> 4)-linked D-galacopyranosyl and alpha-(1 -> 3)-linked D-glucopyranosyl residues substituted at O-6 with glycosyl residues composed of alpha-L-arabinose-(1 -> 4)-alpha-D-glucose (1 -> linked residues. (c) 2005 Elsevier Ltd. All rights reserved.