OBJECTIVE The purpose of this study was to determine the stereospecificity of the apical oligopeptide transporter(s) for the stereoisomers of Val-Val-Val and to determine whether the interaction of these molecules with this transporter(s) could be correlated with their cellular uptake and/or transepithelial transport. METHODS The interactions of these stereoisomers with this transporter(s) were evaluated by determining their ability to inhibit [3H]cephalexin uptake into Caco-2 cells. The metabolism of these stereoisomers was determined in a homogenate of Caco-2 cells and in the apical bathing solution over Caco-2 cell monolayers. The cellular uptake and transepithelial transport properties of these stereoisomers were studied using the Caco-2 cell monolayers. RESULTS The L-L-L tripeptide was totally degraded within 1 h in the Caco-2 cell homogenate and within 2 h when applied to the apical side of a Caco-2 cell monolayer. In contrast, 36.7 +/- 1.3% and 69.7 +/- 0.9% of L-Val-L-Val-D-Val remained after 2 h in the cell homogenate and in the apical bathing solution, respectively. The other six stereoisomers of Val-Val-Val were completely stable in the Caco-2 cell homogenate. Five of the stereoisomers (L-L-L, L-L-D, L-D-L, D-L-L, D-D-L) significantly inhibited the cellular uptake of [3H]cephalexin (91%, 62%, 14%, 45%, 16%, respectively). The other stereoisomers had no effect on the [3H]cephalexin uptake. When the cellular uptake of the stereoisomers was determined, the D-L-L and L-D-L tripeptides showed the highest intracellular concentrations (1.32 +/- 0.25 and 0.62 +/- 0.20 nmol/mg protein after a 2-h incubation, respectively). In contrast, the intracellular concentrations of the other stereoisomers were less than 0.1 nmol/mg protein. Moreover, the cellular uptake of the D-L-L and L-D-L tripeptides was inhibited by Gly-Pro by 82% and 68%, respectively, whereas Gly-Pro showed moderate to no inhibitory effect on the cellular uptake of the other stereoisomers. The permeability coefficients of the stereoisomers across the Caco-2 cell monolayers were very low (1.8 to 3.1 x 10(-7) cm/sec) and almost identical. Gly-Pro had no effect on their transepithelial transport. CONCLUSIONS These results suggest that the interaction of the Val-Val-Val stereoisomers with the apical oligopeptide transporter(s) could be a good predictor of their cellular uptake. However, since the major transepithelial transport mechanism of Val-Val-Val stereoisomers is passive diffusion via the paracellular route, the binding of these molecules to the oligopeptide transporter(s) is not a good predictor of their transepithelial transport. It appears that the stereochemical requirements for the transporter that mediates permeation of the peptide across the basolateral membrane may be different from the requirements for the apical transporter that mediates cellular uptake.