The characterization of multilayers from tri-O-isopentyl cellulose assembled by the Langmuir-Blodgett technique is described. The analysis of ultrathin films by X-ray reflection confirmed a homogeneous overall film quality but did not show any Bragg reflection. However, investigation of a superstructure in terms of an alternating multilayer sample from deuterated and nondeuterated isopentylcellulose ethers by neutron reflection revealed a perfectly periodic layer structure. The influence of the degree of substitution of the isopentylcellulose ethers on the formation and structure of multilayers was investigated as well. Multilayers of partially substituted isopentylcellulose exhibit a periodical film structure composed of double layers. The perfection of the films is not influenced by the residual OH functions. The in-plane structure of tri-O-isopentylcellulose multilayers was investigated by electron diffraction in transmission geometry. The cellulose ether forms a helix of 3-fold symmetry, and the helix axes are preferentially oriented parallel to the dipping direction. Upon annealing a three-dimensional crystalline film of layered structure is obtained in which the chains are packed in a monoclinic unit cell. The very small diameter of the cellulose derivatives of less than 10 A/molecule in the layered superstructures and the observation that very perfect and homogeneously oriented multilayers are obtained by the Langmuir-Blodgett technique augment the toolbox of molecular architects interested in the rational construction of supramolecular architectures.