The crystal and molecular structure of a high-temperature polymorph of chitosan was determined by a constrained linked-atom least-squares refinement utilizing intensities derived from electron diffraction data and stereochemical restraints. In this polymorph, the chitosan chains crystallized in an orthorhombic P2(1)2(1)2(1) space group with cell parameters a = 0.807 Nn, b = 0.844 nm, and c (chain axis) = 1.034 nm. There were two antiparallel chains per unit cell and no water of crystallization. The best projected model obtained using the base plane data coupled with stereochemical refinement gave R = 13.3%. With the three-dimensional diffraction set data, the best model had R values of R = 25.1%, R" = 17.2%. In this model, the chitosan molecule adopts a 2-fold helical conformation with Phi = -92.7 degrees, psi = -148.8 degrees, tau = 118 degrees, and the hydroxymethyl groups, O6, arranged in an approximately gt mode. The chitosan molecules are stabilized by two intramolecular hydrogen bonds, a strong one between O5’ and O3 and a weaker one between O5’ and O6. In addition, the crystalline structure is held together by a network of intermolecular hydrogen bonds, linking the nitrogen atoms, N2, to ring oxygen atoms, O5, of an adjacent antiparallel molecule and also to O6 of an adjacent parallel chain along the alpha axis.