Oriented multilayers may be prepared by drying phospholipid dispersions on a glass slide. They provide a convenient model with which to examine motion and orientation in lipid bilayer structures. The lecithin multilayers were examined using a series of four spin labels, consisting of nitroxide free radicals bonded to the 5, 7, 12 or 16 position of stearic acid. Accurate electron spin resonance tensor elements needed to interpret the data were obtained using the nitroxide 2-doxylpropane (the 4′, 4′-dimethyloxazolidine- N-oxyl derivative of acetone) oriented in host single crystals of 2,2,4,4-tetramethylcyclobutanedione. In the multilayers, all four lipid spin labels were found to orient with their long axes perpendicular to the glass slide, but there is a pronounced systematic variation in anisotropy, dependent on the positional isomer used. The anisotropy of the electron spin resonance spectra was measured and tabulated for various degrees of hydration of the lecithin multilayers. Changes in segmental regions were examined by utilizing temperature to change the motion. The splittings can be accounted for in terms of restricted anisotropic motion, and computer simulations based on restricted anisotropic motion and a Gaussian distribution of orientations are in good agreement with the experimental electron spin resonance spectra. The molecular motion increases as the label is moved further from the carboxylend, and for all four spin labels studied, raising the relative humidity increases the molecular motion of the labels in the lecithin multilayers. The effects of hydration are most pronounced when the label is near the carboxyl group of the stearic acid probe. Relative regional differences in motion along the lipid chains tend to be maintained over a wide temperature range.