Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin produced in the K cells of the intestine and secreted into the circulating blood following ingestion of carbohydrate- and fat-containing meals. GIP contributes to the regulation of postprandial insulin secretion and is essential for normal glucose tolerance. We have established a method of assaying GIP in response to nutrients using the intestinal lymph fistula model. Administration of Ensure, a mixed-nutrient liquid meal, stimulated a significant increase in intestinal lymphatic GIP levels that were approximately threefold those of portal plasma. Following the meal, lymph GIP peaked at 60 min (P < 0.001) and remained elevated for 4 h. Intraduodenal infusions of isocaloric and isovolumetric lipid emulsions or glucose polymer induced lymph GIP concentrations that were four and seven times the basal levels, respectively. The combination of glucose plus lipid caused an even greater increase of lymph GIP than either nutrient alone. In summary, these findings demonstrated that intestinal lymph contains high concentrations of GIP that respond to both enteral carbohydrate and fat absorption. The change in lymphatic GIP concentration is greater than the change observed in the portal blood. These studies allow the detection of GIP levels at which they exert their local physiological actions. The combination of glucose and lipid has a potentiating effect in the stimulation of GIP secretion. We conclude from these studies that the lymph fistula rat is a novel approach to study in vivo GIP secretion in response to nutrient feeding in conscious rats.