Sulfonylurea hypoglycemic agents have interindividual variability in the gastrointestinal absorption rate. However, the absorption mechanism at the intestinal epithelium has not yet been clarified. To elucidate contribution of the specific mechanism for transepithelial transport of sulfonylureas, the apical-to-basolateral and basolateral-to-apical transport studies of tolbutamide were carried out using Caco-2 cell monolayers cultured on the polycarbonate membrane. The transported amounts of the substrate were measured by HPLC to estimate the apparent permeability coefficients (P(app)). In the apical-to-basolateral flux, the transport activity of tolbutamide was facilitated when the pH of the apical medium was more acidic than the basolateral one. ATP-depletion decreased the P(app) of tolbutamide. The kinetic analysis of the permeation rate indicated that the saturable process largely contributed to the tolbutamide flux. The P(app) of tolbutamide was lowered by an ionophore and monocarboxylic acids, while dicarboxylic acids and the inhibitor for the anion exchanger had no effect. In addition, mutual inhibition with benzoic acid was observed in transepithelial transport of tolbutamide. On the other hand, the permeation rate of tolbutamide from the basolateral to apical side was concentration-independent and neither affected by metabolic inhibitors, probenecid nor inhibitors for P-glycoprotein. In conclusion, these results suggest that apical-to-basolateral transport of tolbutamide across the Caco-2 cell monolayers is mediated by the pH-dependent specific system, presumably shared with other organic anions such as benzoic acid.