Purpose. To compare the permeability characteristics of HT29-18-C1 colonic epithelial cell line with Caco-2, an established model of intestinal drug transport. Methods. Cell lines were grown as epithelial monolayers. Permeability was measured over a range of transepithelial electrical resistance (R(t)) using a group of drug compounds. Results. HT29-18-C1 develop R(t) slowly when grown in culture, allowing permeability to be measured over a wide range (80-600 Ω · cm2). In contrast, Caco-2 monolayers rapidly develop R(t) of ~300 Ω · cm2 and require Ca2+-chelation to generate R(t) equivalent to human intestine (60-120 Ω · cm2). Permeability of atenolol, ranitidine, cimetidine, hydrochlorothiazide and mannitol across HT29-18-C1 decreased 4-5 fold as R(t) developed from 100-300 Ω · cm2 indicating they permeate via the paracellular route. In contrast, ondansetron showed no difference in permeability with changing R(t) consistent with transcellular permeation. Permeability profiles across low R(t) HT29-18-C1 and pulse EGTA-treated Caco-2 monolayers were the same for all 5 paracellular drugs suggesting that transient Ca2+ removal does not alter selectivity of the tight junctions. Permeabilities of cimetidine, hydrochlorothiazide and atenolol across 100 Ω · cm2 HT29-18-C1 monolayers reflect more closely those reported for the human ileum in vivo than did mature Caco-2 monolayers. Conclusions. HT29-18-C1 monolayers can be used to study drug permeability at R(t) values similar to human intestine without the need for Ca2+ chelation. As such, they offer a useful alternative to Caco-2 for modelling intestinal drug absorption.