The localization and transport characteristics of dipeptide transporters of the intestinal epithelial cell line Caco-2 were examined by measuring the intracellular accumulation and transcellular flux of Bestatin, a dipeptide-like anticancer agent. When added to the apical surface of Caco-2 monolayers grown on microporous membrane filters, Bestatin was accumulated in the cells and was transported unidirectionally to the basolateral side. The cellular uptake of Bestatin from the basolateral as well as from the apical surface was inhibited by excess dipeptides. Bestatin accumulation from the apical surface was dependent on the pH of the incubation medium with an optimal pH of 6.0, whereas uptake from the basolateral surface was insensitive to the medium pH. Kinetic parameters for Bestatin uptake also indicated that the basolateral and apical dipeptide transporters could be distinguished from each other. A sulfhydryl reagent, p-chloromercuribenzene sulfonate, inhibited Bestatin accumulation from both surfaces, although the inhibitory effect on the basolateral transport was greater than that on the apical transport. These findings suggest not only that dipeptide transporters exist on both the apical and basolateral membranes of Caco-2 cells but also that the basolateral dipeptide transporter is distinct from the apical H(+)-dipeptide cotransporter.