The chain conformation of a beta-glucan extracted from black fungus (BFP) was studied by static/dynamic light scattering, viscometry, atomic force microscopy (AFM), and molecular dynamics (MD) simulation. The Mark-Houwink equation and the relationship between M-w and R-g of BFP in water at 25 degrees C were determined to be [eta] = 1.78 X 10(-7)M(w)(1.6) and R-g = 5 x 10(-4)M(w)(0.9), and the molar mass per unit contour length (M-L) and the persistence length (q) were 2724 +/- 276 nm(-1) and 230 +/- 30 nm, respectively, indicating triple-helix conformation. Moreover, the stiff-chain lengths of the BFP fractions were visualized with AFM images, and their M-L values were estimated to give a mean of 2212 nm(-1), consistent with the above. Importantly, MD simulation confirmed that the triple helix was the most stable conformation of BFP. We identified, for the first time, the triple-helix chain conformation of BFP and also offered an alternative method for the characterization of the rigid macromolecules.