A defective epidermal permeability barrier (EPB) in premature birth remains a leading cause of neonatal death as a result of its associated complications, which include poor temperature stability, infection by micro-organisms through the skin, and the outflow of water. Despite its importance in survival, the mechanisms involved in the formation and maintenance of the EPB are not well understood. To address the possibility that claudins, a new superfamily of tight junctional molecules, are involved, we engineered transgenic mice with claudin 6 (Cldn6) overexpressed via the involucrin (Inv) promoter. Interestingly, the Inv-Cldn6 transgenic animals die within 2 days of birth, apparently due to the lack of an intact EPB as evidenced by increased water loss and the penetration of X-gal through the skin. Barrier dysfunction was manifested biochemically by the aberrant expression of late epidermal differentiation markers, including K1, filaggrin, loricrin, transglutaminase 3, involucrin, repetin, members of the SPRR family and the transcriptional regulator Klf4. The overall claudin profile of the epidermis was also modified. Our data suggest that repetin and SPRR1A and 2A are downregulated in response to the downregulation of Klf4 in the transgenic animals, which would contribute to decreased protein crossbridging leading to fragile, defective cornified envelopes. These results provide new insights into the role of claudin 6 in epithelial differentiation and EPB formation. In addition, the epidermal phenotype of these transgenic mice, which is very reminiscent of that in pre-term infant skin, suggest that they will be an important and novel model for studies on human premature EPB-related morbidity.