The stratum corneum of human skin is responsible for maintaining the epidermal permeability barrier, We have developed a bilayered skin culture (SC) which forms a corneum 35 +/- 1 cell layers thick 21 days after being raised to the air-liquid (A/L) interface. By the 7th day after raising to the A/L interface the corneocytes were irregularly shaped and had cross-sectional areas (CSA) of greater than or equal to 300 mu m(2). By the 21st day the corneocytes had assumed polygonal shapes and had a CSA (100-250 mu m(2)) similar to that of human foreskin. The total lipid (TL) content of the corneum averaged 5-7% of the lyophilized weight. Ceramide content increased from 20% of TL at day 7 of A/L interface culture to 30% at dag 21. Triglycerides decreased from 43% to 17% of TL during the same period. Free fatty acids comprised 5.5% of TL at day 21 of A/L interface culture. The intercorneocyte spaces contained stacks of lipid lamellae. However, the stacks lacked the Landmann unit repeat. Abnormal lamellar structures were observed in both the intra- and extracorneocyte spaces. Transepidermal mater loss (TEWL) was > 4mg/cm(2) per h throughout the culture period. Lipid supplementation of the culture medium and culturing in a low humidity environment improved barrier function by 50%. However, the effects mere not additive. The SC developed a near-normal corneum, but did not achieve barrier competence, due at least partially to abnormalities in lipid composition and organization. Improvement of barrier function with lipid supplementation or low humidity indicates that modifications of the culture environment may facilitate the SC in assembling a permeability barrier equivalent to human skin.