The molecular and crystal structure of the anhydrous form of chitosan, a (1-->4)-linked 2-amino-2-deoxy-beta-D-glucan, has been determined by combined X-ray diffraction analysis and stereochemical model refinement. Chitosan chains crystallize in an orthorhombic unit cell with dimensions a = 0.828 nm, b = 0.862 nm, and c (fiber axis) = 1.043 nm. Systematic absences are consistent with the space group P2(1)2(1)2(1). The unit cell comprises four glucosamine residues, giving a density of 1.52 g/cm(3). The X-ray diffraction pattern was recorded on the imaging plate, and an intensity of each reflection spot was estimated by two-dimensional measurement and subsequent background removal. Chain conformation is an extended a-fold helix stabilized by intramolecular O3...O5 hydrogen bonds, and an O6 atom is rotated at near gt position. Two chains pass through the unit cell with an antiparallel packing arrangement. Intermolecular N2...O6 hydrogen bonds contribute to the three-dimensional stabilizing of the crystal structure. The reliability of the structure analysis is indicated by the X-ray residual R = 0.175 and R" = 0.178 against the observed 38 hkl’s. Some significant similarities are observed between the anhydrous crystal structures of the present chitosan and (1-->4)-beta-D-mannan.