The characteristics of glycyl-L-leucine uptake have been studied using normal and papain-treated brush border membrane vesicles prepared from mouse small intestine. Our results show that: (1) glycyl-L-leucine is taken up into an osmotically reactive intravesicular space; (2) intact peptide transport can be studied up to 5 min without interference of hydrolytic events when using papain-treated vesicles; (3) the time course curves of glycyl-L-leucine uptake by normal and papain-treated vesicles are identical whatever the composition of the media (mannitol, NaSCN or KSCN) and do not show any overshoot phenomenon in the presence of an electrochemical gradient of Na+ (extravesicular greater than intravesicular); (4) a linear relationship exists between initial rates of dipeptide uptake and peptide concentrations; (5) peptide uptake is weakly inhibited by other di- and tri-peptides at a 60 mM concentration. We can conclude that intact peptide transport occurs down a concentration gradient by a non-Na+-dependent process and that passive and facilitated diffusion mechanisms, the latter either by a high affinity-low capacity system or a low affinity-high capacity system, are involved in this transport. It also appears that gamma-glutamyltransferase and the gamma-glutamyl cycle are not involved in peptide absorption.