The effect of in vivo infusion of the peptide hormone glucagon-like peptide 2 (GLP-2) on glucose transport across the rat jejunal brush-border membrane (BBM) was assessed using isolated membrane vesicles. A 2-h infusion of GLP-2 produced a marked acceleration of sodium-dependent glucose uptake into BBM vesicles with a significant overshoot. There was no change in vesicle space or permeability resulting from the hormone infusion. Kinetic analysis showed this stimulation to be the result of a three-fold increase in the maximal rate of transport, with no consistent change in the affinity constant (Km). The time course of this response showed that the effect was observable, but smaller, after only 30 min of hormone infusion and was maximal after 1 h. Sodium-dependent phloridzin binding to the membrane vesicles showed a parallel increase in maximal binding after 1 and 2 h of hormone infusion. Western blotting showed a similar increase in sodium-dependent glucose transporter 1 (SGLT-1) abundance. The effect of GLP-2 could be blocked by luminal brefeldin A or wortmannin. These results indicate that GLP-2 is able to induce trafficking of SGLT-1 from an intracellular pool into the BBM within 60 min and that phosphoinositol 3-kinase may well be involved in the intracellular signaling pathway in this response.