The epidermis (including stratum corneum) of human keratinocytes cultured at the air-liquid interface attached to an appropriate substrate shows a morphology closely mimicking that of its in vivo counterpart. In spite of the histologic similarities, the barrier function seems to be impaired. The aim of the present study was to characterize development and structure of the epidermal permeability barrier in two human skin recombinants using electron microscopy (including ruthenium tetroxide-post fixation technique) and analysis of lipid composition. The epidermis was reconstructed by growing human keratinocytes either on de-epidermized dermis or on a bovine collagen-containing matrix with active fibroblasts (Living Skin Equivalent). Ultrastructurally both culture systems showed a) an abnormal lamellar body delivery system, b) disturbance of transformation into lamellar lipid bilayers, c) an impaired structural organization and distribution of the epidermal lipids in the intercellular spaces. In either of the systems used, prolongation of the culture period did not induce any significant improvement in the stratum corneum lipid organization. Whereas the Living Skin Equivalent showed only sparse lamellar bodies, the number of lamellar bodies in the human keratinocyte culture on de-epidermized dermis grown in regular medium seemed to be comparable to native skin. Contrary to the Living Skin Equivalent, the keratinocyte culture on de-epidermized dermis contained a higher number of intracorneocytic lipid droplets correlating with a higher triglyceride content in the lipid analyses. By reconstructing the keratinocyte culture on de-epidermized dermis with the same medium as used for the Living Skin Equivalent, both lipid composition (lower triglyceride, higher ceramide contents) and structural organization were improved, and regular lamellar lipid bilayers comparable to those of native skin appeared