After the ingestion of nutrients, secretion of the incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) by the enteroendocrine cells increases rapidly. Previous studies have shown that oral ingestion of fat stimulates secretion of both incretins; however, it is unclear whether there is a dose-dependent relationship between the amount of lipid ingested and the secretion of the hormones in vivo. Recently, we found a higher concentration of the incretin hormones in intestinal lymph than in peripheral or portal plasma. We therefore used the lymph fistula rat model to test for a dose-dependent relationship between the secretion of GIP and GLP-1 and dietary lipid. Under isoflurane anesthesia, the major mesenteric lymphatic duct of male Sprague-Dawley rats was cannulated. Each animal received a single, intraduodenal bolus of saline or varying amounts of the fat emulsion Liposyn II (0.275, 0.55, 1.1, 2.2, and 4.4 kcal). Lymph was continuously collected for 3 h and analyzed for triglyceride, GIP, and GLP-1 content. In response to increasing lipid calories, secretion of triglyceride, GIP, and GLP-1 into lymph increased dose dependently. Interestingly, the response to changes in intraluminal lipid content was greater in GLP-1- than in GIP-secreting cells. The different sensitivities of the two cell types to changes in intestinal lipid support the concept that separate mechanisms may underlie lipid-induced GIP and GLP-1 secretion. Furthermore, we speculate that the increased sensitivity of GLP-1 to intestinal lipid content reflects the hormone's role in the ileal brake reflex. As lipid reaches the distal portion of the gut, GLP-1 is secreted in a dose-dependent manner to reduce intestinal motility and enhance proximal fat absorption.