The H+-coupled transporter, peptide transporter 1 (PepT1), is responsible for the uptake of dietary di- and tripeptides in the intestine. Using an in vivo continuously perfused gut loop model in Yucatan miniature pigs, we measured dipeptide disappearance from four 10 cm segments placed at equidistant sites along the length of the small intestine. Pigs were studied at 1, 2, 3 (suckling) and 6 weeks (post-weaning) postnatal age. Transport capability across the PepT1 transporter was assessed by measuring the disappearance of 3H-glycylsarcosine; real-time RT-PCR was also used to quantify PepT1 mRNA. Each of the regions of intestine studied demonstrated the capacity for dipeptide transport. There were no differences among age groups in transport rates measured in the most proximal intestine segment. Transport of 3H-glycylsarcosine was significantly higher in the ileal section in the youngest age group (1 week) compared with the other the suckling groups; however, all suckling piglet groups demonstrated lower ileal transport compared with the post-weaned pigs. Colonic PepT1 mRNA was maximal in the earliest weeks of development and decreased to its lowest point by week 6. These results suggest that peptide transport in the small intestine may be of importance during the first week of suckling and again with diet transition following weaning.