The epidermal permeability barrier appears to be regulated primarily by the lamellar arrangement of lipid bilayers between coneocytes of the stratum corneum and presents a significant barrier to the transdermal delivery of drugs. The aim of the present study was to investigate the effects of oleic acid on the ultrastructure of stratum corneum lipids in rat skin. Wistar rats were treated topically with 10% oleic acid/propylene glycol for 2 h, the structure of stratum corneum was examined by electron microscopy using osmium tetroxide or ruthenium tetroxide postfixation, and the epidermal barrier function was evaluated in a lanthanum tracer study. Ultrastructural examination revealed that there was a marked alteration in the stratum corneum and the tracer penetrated into the intercellular spaces of the stratum corneum after application of oleic acid. These results suggest that ruthenium tetroxide postfixation is a powerful tool for the study of the stratum corneum lipid structure. Oleic acid might increase the epidermal permeability through a mechanism involving the perturbation of stratum corneum lipid bilayers and lacunae formation to enhance transdermal drug delivery.