The objective of this study was to elucidate the structural features of the stereoisomers of Val-Val and Val-Val-Val that afford optimal binding affinity for the apical oligopeptide transporter in human intestinal Caco-2 cells. Three-dimensional conformations of cephalexin and Val stereoisomers were optimized using Chem-X molecular modeling software. Molecular features associated with the optimized conformations of the Val stereoisomers were analyzed to identify potential relationships with their binding affinities for the apical oligopeptide transporter. For Val-Val stereoisomers, the distance between the N-terminal amino group and the C-terminal carboxyl group, d(N1-C7), was found to have a linear relationship with their binding affinities at the 95% confidence level. For Val-Val-Val stereoisomers, three molecular features were found to have linear relationships with their binding affinities at the 95% confidence level. These features included: a) the distance between the N-terminal amino group and the C-terminal carboxyl group, d(N1-C11); b) the distance between the N-terminal amino group and the second peptide bond, d(N1-N9); and c) the molecular dipole moment. Principal component analysis on all molecular features of Val-Val-Val stereoisomers identified three components that accounted for 90% of the variance. A linear model built with these three components by multiple linear regression adequately described the binding affinities (r2 = 0.90). Results from the current study suggest that the distance between the N-terminal amino group and the C-terminal carboxyl group is important for interaction with the apical oligopeptide transporter in Caco-2 cells. In addition, the binding affinities of the Val-Val-Val stereoisomers appear to be influenced by additional factors, including the position of the second peptide bond and the molecular dipole moment.