To investigate the mechanism responsible for the intestinal absorption of a lipophilic organic cation, quinidine, we performed a pharmacokinetic analysis of transcellular transport across Caco-2 cell monolayers grown on a porous membrane. Basolateral-to-apical transport of the drug was almost constant in the concentration range of 100 nM—100 μM. Transcellular transport was greater in the apical-to-basolateral direction than in the opposite direction. Apical-to-basolateral transport was greater at a concentration of 100 μM than 100 nM. The calculated influx clearance value of the apical membrane was much greater than the other influx/efflux clearance values of cell membranes, and was 5.6-fold the influx clearance value of the basolateral membrane at the drug concentration of 100 μM. We also investigated the uptake of quinidine at the apical membrane of Caco-2 cells grown on plastic dishes. The uptake was markedly increased by alkalization of the apical medium at 37 °C, and was decreased at low temperature (4 °C). In addition, it was inhibited by diphenhydramine and levofloxacin, but not by carvedilol, rifamycin SV, or L-carnitine. These findings indicated that the influx at the apical membrane was the direction-determining step in the transcellular transport of quinidine across Caco-2 cell monolayers, and that some specific transport system was involved in this influx.