The transepithelial transport and uptake of chloroquine were studied in cultured human intestinal Caco-2 cell layers, to investigate whether a specific mechanism facilitates the flux of chloroquine. Due to ionization of chloroquine at the pH of the intestinal lumen, the fraction of the neutral form, which is required for partitioning into biological membranes, is very low, while oral bioavailability has been reported to be nearly complete. Several observations, such as concentration-dependent uptake and temperature-dependent transepithelial flux, suggest the presence of carrier mediated transport. However, alternative mechanisms may be invoked to explain these observations. It is suggested that concentration dependence can originate from ion-trapping in acidic compartments of the cell or non-specific binding to cell components, while temperature-dependent transport can, at least partly, be explained by the temperature dependence of the acid dissociation constants of chloroquine. No differences were observed in the transepithelial flux of the enantiomers of chloroquine. pH-dependent uptake as well as pH-dependent transepithelial transport suggest that the translocation of chloroquine occurs according to the fraction of neutral molecules. From the data obtained in this study, it is concluded that chloroquine crosses the gastrointestinal barrier by passive diffusion. The extensive area of the gastrointestinal tract probably compensates for the low fraction of the neutral molecule. An interesting finding of this study was the concentration-dependent increase in transepithelial electrical resistance across monolayers incubated with chloroquine at the apical side.