A simple, reliable, and user friendly system was established to cultivate Caco-2 cell monolayer for epithelial transport studies. After an initial growth period of 1 week in a CO 2 incubator, Caco-2 cells were cultivated in an automated continuous perfusion system (Minucells and Minutissue, Germany). Medium was constantly renewed at the apical and basal side of the monolayers, which resulted in a continuous supply of nutrients as well as in a continuous removal of metabolite wastes. The monolayers obtained with the new perfusion culture system were evaluated to estimate the passive transport properties of a series of model compounds. The results produced were compared to those of monolayers obtained with the standard 21-day system. The integrity of cell monolayers was checked by measuring transepithelial electrical resistance (TEER) and by the transport of the paracellular leakage marker sodium fluorescein. The results of confocal microscopy as well as TEER measurements indicated the formation of a monolayer on various support filters. The growth and differentiation of Caco-2 cells were highly dependent upon the individual support filters and extracellular matrix proteins used for Caco-2 attachment. The permeability coefficients of several model compounds across Caco-2 cells obtained with the perfusion system were approximately two-fold higher than those obtained using the traditional 21-day Snapwell-based cultures. A good correlation was found between the transport of passively diffused drugs across Caco-2 monolayers differentiated in the perfusion system and the transport according to the standard method. The rank ordering of high permeable model compounds tested through Caco-2 monolayers, differentiated in a perfusion system, was similar to the standard 21day culture method.