The barrier function of the skin resides in the stratum corneum (SC). This outermost layer consists of protein-rich corneocytes and lipid-rich intercellular domains. These domains form the rate-limiting step for transepidermal water loss and the penetration of substances from the environment. To study the nature of the barrier function, stratum corneum lipid models have been examined with wide-angle X-ray diffraction. A disadvantage of this technique is that it requires bulk quantities of lipid and thus information on variations in the lateral packing cannot be obtained in the microm-range. To the best of our knowledge, this is the first study in which electron diffraction is applied on SC lipid model systems. Using this technique, local structural information was obtained about mixtures prepared from isolated pig ceramides, cholesterol, and long-chain free fatty acids. It appeared that addition of free fatty acids caused a transition from a hexagonal to an orthorhombic packing and that electron diffraction can be applied to distinguish between these two lattices. The results are in good agreement with wide-angle X-ray diffraction data and suggest that application of electron diffraction in skin studies can provide new information on the lipid organization in well-defined areas of the stratum corneum.