The intercellular lipid lamellae of the stratum corneum (SC) is believed to provide the permeability barrier of the epidermis. Previous functional studies have demonstrated an increase in the transepidermal water loss (TEWL) after long-term use of topical corticosteroids (TCS); however, direct morphological confirmation of this barrier abnormality is still lacking. The aim of this study was to determine whether any abnormality could be detected in the structure of the SC intercellular lipid lamellae in patients after long-term TCS. Atrophic skin and untreated normal skin of 10 patients after long-term TCS were examined by transmission electron microscopy using ruthenium tetroxide-fixed tissue for the multilamellar lipid sheets of SC, and oil red O stain for neutral lipids of the SC. Layers of the SC were evaluated by 0.1% methylene blue stain after alkaline expansion, and TEWL was measured by Evaporimeter EP1. The TCS-treated atrophic skin had fewer layers of horny cells, mean 9.4 layers, than the normal control skin, 18 layers (P < 0.001) and increased TEWL of 21.3 g/m2 compared with the control skin TEWL of 6.7 g/m2 (P < 0.01). The mean neutral lipid content of the SC was also significantly lower (P < 0.001). Moreover, ultrastructural studies revealed a marked decrease in both the numbers of intercellular lipid lamellae of SC and membrane-coating granules of stratum granulosum in the atrophic skin. These results suggest that the diminution in the SC intercellular lipid lamellae and SC cell layers play an important part in the pathogenesis of barrier dysfunction after long-term use of TCS.