Despite the importance of intercellular lamellar bilayers for stratum corneum (SC) barrier function, knowledge about the structure of these bilayers is limited due to their poor visualization and/or retention. Whereas substitution of ruthenium tetroxide (RuO₄) for osmium tetroxide fixation provides clear images of these bilayers, the usefulness of RuO₄ has been limited by its slow penetration and cytotoxicity. Utilizing a new fixation protocol for RuO₄, we obtained clear images of lamellar domains at all levels of murine SC. Computer-aided image reconstructions demonstrated a lamellar spacing of 129 ± 2 Å, which agreed with x-ray diffraction data from parallel, unfixed samples (131 ± 2 Å), a spacing not affected by hydration. Furthermore, novel structures were seen in the intercellular spaces of normal SC. Finally, in murine essential fatty acid deficiency (EFAD), the overall lamellar spacing is comparable to normal [127 ± 7 Å by computer transform vs. 131.9 ± 2 Å (hydrated) and 129.6 ± 2.2 Å (dry) by x-ray diffraction]. Yet, these domains are structurally abnormal, displaying regions with either an excess or absence of lamellae. The new RuO₄ protocol provides quantitative information about SC lamellar dimensions and morphologic abnormalities in bilayer distribution and substructure in EFAD stratum corneum that are not detected by either x-ray diffraction or computer-aided image reconstruction. Thus, the barrier abnormality in EFAD stratum corneum can be ascribed either to focal depletion of lamellae or abnormalities in lamellar substructure.