Keratinocyte monolayers, cultured in immersed conditions, constitute a frequently used in vitro model system to study keratinocytes behaviour in response to environmental assaults. However, monolayers lack the keratinocyte terminal differentiation and the organization of the epidermal tissue, which are observed in vivo. Advancements of in vitro techniques were used to reconstruct three-dimensional equivalents that mimic human epidermis in terms of layering, differentiation and barrier function. Here, we update a published method and illustrate the progressive morphogenesis responsible for in vitro reconstruction. The analysis of cell proliferation, expression of differentiation markers and barrier efficacy demonstrate the excellent similarity of the reconstructed tissue with normal human epidermis. Availability of epidermal tissue during its reconstruction phase in culture appears crucial for studies intending to challenge the barrier function.