Caco-2 cells develop morphologic characteristics of normal enterocytes when grown on plastic dishes or nitrocellulose filters. The purpose of this study was to determine whether Caco-2 cells undergo similar differentiation when grown on Transwell polycarbonate membranes, and to study the suitability of Caco-2 monolayers as an intestinal epithelial transport model system. Transepithelial electrical resistance values after confluence were 173.5 Ω · cm² and remained unchanged through day 17. Permeabilities to the water-soluble fluid-phase markers that do not permeate the membrane, Lucifer yellow CH, [¹⁴C]inulin, [¹⁴C]polyethylene glycol, and [³H] dextran were <0.25% of the administered amount per hour after day 10. Qualitative evaluation of uptake and permeability to horseradish peroxidase confirmed the similarity in uptake and barrier properties between this cell system and the small intestinal epithelial layer. We conclude that Caco-2 cells grown on collagen-coated polycarbonate membranes should represent a valuable transport model system for the small intestinal epithelium.