The aim of this work was to investigate the contribution of the paracellular route to the pH-dependent permeability to cationic drugs in three models expressing different drug permeabilities: hexadecane membranes (HDMs), Caco-2, and 2/4/A1 cell monolayers. The high- and low-permeability drugs alfentanil and cimetidine were used as model drugs. The paracellular permeability was calculated: 1. from the assumption that the ionized form (P(mi)) permeates a cell monolayer only by the paracellular route, and 2. on basis of the pore-restricted diffusion. For both drugs, sigmoidal relationships between membrane permeability and pH were observed in all models. The P(mi) was in excellent agreement with the paracellular permeability of cimetidine in the two cell models, whereas no significant P(mi) of the drugs could be observed in HDM. The results showed that the paracellular route has a significant role in the permeability of small basic hydrophilic drugs, such as cimetidine in leaky, small intestinal-like epithelia such as 2/4/A1. By contrast, in tighter epithelia such as Caco-2 and in artificial membranes such as HDM, the permeability of the ionized forms of the drugs and the paracellular permeability are lower or insignificant, respectively. These findings will have implications in the experimental design and data interpretation of pH-dependent drug transport experiments in cell culture models as well as in artificial membrane models such as HDM and parallel artificial membrane permeability assay (PAMPA).