Water-insoluble polysaccharide (TM3a), extracted from sclerotia of Pleurotus tuber-regium, was identified as a hyperbranched beta-(D)-glucan from the results of one- and two-dimensional NMR and GC-MS analysis. The degree of branching of TM3a is 65.5%. TM3a was fractionated by using a non-solvent addition method into 14 fractions, and its solution properties in 0.25 M LiCl/dimethylsulfoxide (DMSO) solution were studied systematically by using static laser light scattering, dynamic light scattering, and viscometry at 25 degrees C. The dependences among the values of intrinsic viscosity ([eta]), radius of gyration (< S-2 >(1/2)(z)), and hydradynamic radius (R-h) on weight-average molecular weight (M-w) were found as the following: [eta] = 0.46M(w)(0.30 +/- 0.01), < S-2 >(1/2)(z) = 4.79 x 10(-2)M(w)(0.43 +/- 0.04), and R-h = 5.01 x 10(-2)M(w)(0.41 +/- 0.02) in the M-w range from 1.94 x 10(5) to 2.06 x 10(7) for TM3a in a 0.25 M LiCl/DMSO solution at 25 degrees C. The current theory of polymer solution was applied to explain the relationship among the fractal dimension, ratio of geometric to hydrodynamic radius (rho = < S-2 >(1/2)(z)/R-h), and M(w)A(2)/[eta] of TM3a. The results indicated that TM3a existed as a compact chain conformation with a sphere-like structure in LiCl/DMSO solution. Furthermore, by using transmission electron microscopy, we observed directly the spherical molecules with an average diameter of 23.0 +/- 1.8 nm.