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Aryl hydrocarbon receptor nuclear translocator (ARNT), a transcription factor of the Per/AHR/ARNT/Sim family, regulates gene expression in response to environmental stimuli including xenobiotics and hypoxia. To examine its role in the epidermis, the Cre-loxP system was used to disrupt the Arnt gene in a keratinocyte-specific manner. Gene-targeted, newborn mice with almost normal appearance died neonatally of severe dehydration caused by water loss. Histology showed small changes in the architecture of cornified layers, with apparently preserved intercorneocyte lamellar structures responsible for the skin barrier function. In contrast, HPLC/ion-trap mass spectrometry revealed significant alterations in the compositions of ceramides, the major components of the lamellae. The murine epidermal ceramides normally contain 4-sphingenine and 4-hydroxysphinganine. In Arnt-null epidermis, 4-sphingenine was largely replaced by sphinganine and the amounts of ceramides with 4-hydroxysphinganine were greatly decreased, suggesting deficiency of dihydroceramide desaturases that catalyze the formation of both 4-sphingenyl and 4-hydroxysphinganyl moieties. A desaturase isoenzyme, DES-1, prefers desaturation, but DES-2 catalyzes both reactions to a similar extent. Transcript levels of Des-2, but not Des-1, were considerably decreased in cultured keratinocytes from Arnt-null epidermis. These results indicate that proper ceramide compositions through 4-desaturation regulated by ARNT are crucial for maintaining the epidermal barrier function.