Polysaccharide components in hot-water extract of Ganoderma lucidum fruiting bodies were separated and their molecular weights and distribution profiles were measured using high-performance size exclusion chromatography with multi-angle laser light scattering detection (HPSEC-MALLS). Three polysaccharide groups were found in the extract with a weight percentage of 31, 46 and 23%, respectively. The weight-average molecular weights of these groups were 2.08 ¥ 106, 2.3 ¥ 104 and 1.2 ¥ 104 g/mol, respectively. The representative fractions of these 3 groups were obtained using differential precipitation with ethanol at 20, 60 and 80% (v/v) concentrations for further property study. The first group that had the largest molecular size was branched (1→3)-b-D-glucan with single b-Dglucosyl side chains at the O-6 positions, as revealed from sugar composition analysis, aniline blue assay and 1H- and 13C-NMR spectroscopy. (1→3, 1→6)-b-D-glucan was the major active polysaccharide and showed significant tumor necrosis factor-a (TNF-a) releasing stimulation activity from human mononuclear cells (MNC). The glucan was slightly soluble in water at ambient temperature. The second and third groups that showed slight modulating activity consisted of D-glucose, D-galactose and D-mannose at different ratios. Aggregation of polysaccharide molecules was also revealed by HPSEC-MALLS study.