Culturing of normal human keratinocytes at the air-liquid interface results in the formation of fully differentiated epidermis under in vitro conditions. Although the reconstructed epidermis shows a close resemblance to native tissue, there are still some differences in the stratum corneum lipid profile and intercellular lipid organization. As ceramides belong to one of the major stratum corneum lipid classes, the aim of this study was to characterize this fraction in more detail. For this purpose, individual ceramide fractions were isolated by column chromatography and characterized by a combination of nuclear magnetic resonance spectroscopy, high-performance thin-layer chromatography, and gas chromatography. The results of this study show that in both the native and reconstructed human epidermis the extractable ceramide fraction contains, in addition to the well known acylceramides (EOS, EOH), a new acylceramide in which the omega-O-acylhydroxyacid is amide-linked to phytosphingosine (EOP). The same three sphingoid base moieties (S, P, H) are also found in ceramides with amide-linked nonhydroxy and alpha-hydroxyacids. Whereas the same types of ceramides were present in both tissues, some differences in their fatty acid profiles have been found. In reconstructed epidermis the content of linoleic acid in all three acylceramides fraction was significantly lower; the ceramide(NS) fraction was enriched in short fatty acids and the ceramide(AS) fraction was enriched in long chain alpha-hydroxyacids. These differences together with a lower content of free fatty acids may explain the differences between native and reconstructed tissue in stratum corneum lipid organization observed earlier by X-ray diffraction.