The aim of the study was to characterize and assess the maturation process of the barrier function in a reconstituted human epidermis (REp) cultivated in a chemically defined medium. For this purpose histomorphological analysis, percutaneous absorption studies and non-invasive measurements were performed. In order to understand the time course of the barrier development, REp cultivated from the same pool of normal human keratinocytes were harvested and measured after several increasing periods of exposure at the air-liquid interface. From these results we concluded that a maturation period of at least 16 days at the air-liquid interface was required for an optimal barrier function development of REp. At this time point, the permeability of the skin cultures for caffeine (CAF) was 20-25-fold higher than that of normal human skin (NHS) biopsies. Non-invasive measurements of skin water content, transepidermal water loss (TEWL) and pH were achieved after the same period of time. Results showed that the stratum corneum (SC) of REp was slightly more hydrated than that of ex vivo and in vivo, human skin. TEWL was slightly higher through REp than through NHS, and the pH of the REp models was very close to that of in vivo, normal human skin. In the latter part, assuming that this type of model could be routinely used, we quantified over a 1-year period the barrier function variability of this skin culture model between batches. The satisfactory results obtained with a 20% coefficient of variation indicated that this REp model has a consistent and reproducible barrier function. This leads us to suggest that the skin model might be considered an alternative membrane to normal human skin for permeation screening tests.