The aim of this study was to define the permeability characteristics of the parental Caco-2/ATCC cell line and of three clonal lines (Caco-2/TC7, Caco-2/AQ, Caco-2/15) differentiated in serum-supplemented or in serum-free defined medium. The Caco-2 cells were grown in DMEM supplemented with either 10% foetal calf serum or insulin-transferrin-selenium and lipids (cholesterol, palmitic acid, oleic acid) for up to 24 days after seeding on polyethylene terephthalate filter inserts (1 microm pore diameter). The permeability of the cell monolayer was assessed by measuring trans-epithelial electrical resistance (TEER) and the apparent permeability (Papp) of the extracellular marker mannitol during differentiation from day 6 until day 24. In all lines TEER values increased during differentiation reaching a plateau value around day 15 after seeding, while the Papp for mannitol decreased sharply around day 8 and levelled off thereafter. Substantial differences were observed in the maximal TEER values achieved during differentiation in the four lines examined (Caco-2/TC7 <Caco-2/ATCC<Caco-2/AQ<Caco-2/15), while the Papp for mannitol, upon differentiation, was not significantly different in all four lines. Media composition (serum-supplemented vs. defined medium) did not generally affect the TEER (with the exception of the Caco-2/ATCC). Conversely, the presence of serum in the medium consistently lowered and stabilised Papp values as compared with defined medium.