Stratum corneum lipids have been found to be an important determinant involved in the water-holding function of the stratum corneum. In order to further elucidate the significance of stratum corneum lipids in the water-holding properties, the hydration behavior of the stratum corneum following depletion and replenishment of the lipids has been studied, using an isolated human forearm stratum corneum sheet, by differential scanning calorimetry parallel to ultrastructural changes of intercellular lipids in the stratum corneum. Extraction of the stratum corneum sheet with acetone/ether (1/1) decreased the bound-water content from 33.3% to 19.7%, where the melting temperature of ice remained constant. Further extraction with water, which released a large amount of water-soluble materials such as amino acids, did not change the bound-water content, but definitely raised the melting temperature of ice. The application of the stratum corneum lipids, which were solubilized in squalane containing 1% alpha-monomethyl heptadecyl glyceryl ether to the lipid-depleted stratum corneum sheet, caused a significant recovery of bound-water content to the previous, almost normal level. In accordance with the hydration behavior, electron microscopic analysis of the acetone/ether-treated stratum corneum sheet revealed selective depletion of lipids from the intercellular spaces, accompanied by a marked disruption of multiple lamellar structures. In contrast, the application of stratum corneum lipids into the lipid-depleted stratum corneum sheet resulted in the restoration of the lamellar structure between the stratum corneum cells. These findings strongly suggest that stratum corneum lipids serve a water-holding function through the formation of lamellar structures within the stratum corneum.