Glucagon-like peptide-1 (GLP-1) is an important incretin produced in the L cells of the intestine. It is essential in the regulation of insulin secretion and glucose homeostasis. Systemic GLP-1 concentrations are typically low in rodents so it can be difficult to assay physiologic levels or detect changes in response to nutrients. We have established a method of assaying GLP-1 in response to nutrients using the intestinal lymph fistula model. Intraduodenal infusion of Intralipid (4.43 Kcal/3 ml) induced a significant increase of lymphatic GLP-1 concentration compared to saline control at the peak of 30 min. (P < 0.001). Isocaloric and isovolumetric treatment with dextrin, a glucose polymer, also caused a significant 4-fold increase in peak concentration at 60 min (P = 0.001). These findings indicate that intestinal lymph contains high concentrations of postprandial GLP-1. Second, they reveal that GLP-1 secretion into lymph occurs in response to both enteral carbohydrate and fat, but the response to dextrin occurs later than to Intralipid with peak times at 60 and 30 min, respectively. Third, the combination of Intralipid plus dextrin demonstrated an additive effect in the stimulation of GLP-1 with peak at 30 min. These results indicate that assessment of levels in lymph is a novel and powerful means of studying the secretion of GLP-1 and potentially other gastrointestinal hormones in vivo. Furthermore, the lymph fistula rat model provides insight into the gut hormone concentrations to which the neurons and cells in the lamina propria of the gut are likely exposed.