A fully differentiated epithelium having the features of epidermis was obtained in vitro by culturing second-passage normal human keratinocytes (NHK) in the chemically defined medium MCDB 153 on inert filter substrates at the air-liquid interface for 14 d. Vertical sections stained for histology and indirect immunofluorescence studies show a correct stratification and expression of differentiation markers. The presence of desmosomes, keratohyalin granules, and lamellar granules, and the formation of a more than ten-layers stratum corneum was evidenced by electron microscopy. Moreover, lipids typical for differentiated epidermis were present in these cultures, including ceramides, which are thought to be responsible for the relative impermeability of the stratum corneum. Under our culture conditions, i.e., in defined medium and at the air-liquid interface, the use of de-epidermized dermis as a substrate did not stimulate keratinocyte differentiation more than acetate cellulose or polycarbonate filter membrane substrates. The obtaining of a well-differentiated epidermis grown in vitro on inert filters in a chemically defined medium should be useful as a standard system for studying epidermal differentiation, re-epidermization, cytotoxicity, epidermal permeation, and transepidermal drug delivery.