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Notes: Abstract: Cornified envelopes (CEs), rigid and insoluble structures in the stratum corneum, which are assembled by crosslinking of several precursor proteins by transglutaminases, provide a hydrophobic foundation for barrier function; omega-hydroxyceramides are covalently attached to the outer surface of CE components, and onto this hydrophobic assembly, lamellar layers of intercellular lipids are organized. Morphologically irregular, fragile CEs are found in the deep layer of the stratum corneum or in certain disorders, such as psoriasis, whereas most CEs from healthy subjects are rigid and polygonal. We have established a staining method to characterize such fragile CEs as immature and less hydrophobic CEs, and employed it to examine regional differences in the properties of CEs, especially in relation to the barrier function of the skin. CEs from the outermost stratum corneum of the trunk and extremities of healthy subjects were relatively uniform in morphology with larger shape, and were homogeneous in hydrophobicity as judged from the use of an environment-sensitive fluorescent dye, Nile red. However, CEs from the face were strikingly heterogeneous, and consisted of both rigid and fragile CEs. Rigid CEs were Nile red-positive and little stained by anti-involucrin. In contrast, fragile CEs were Nile red-negative but strongly stained with anti-involucrin, as detected by indirect immunofluorescence. Thus, CEs from the face were stained with Nile red or involucrin in a mutually exclusive manner. Fragile CEs were stained with antibodies against other CE components, including loricrin, envoplakin, filaggrin, and isopeptides. Such fragile, involucrin-positive CEs were detected not only in the face, but also in the deep layer of the stratum corneum of the arm. In addition, experimental barrier disruption resulted in the appearance of involucrin-positive CEs in the outermost stratum corneum. These results suggest that involucrin-positive, fragile CEs are immature and less hydrophobic, and that their occurrence is closely related to impairment of the barrier function of the skin