Following the ingestion of nutrients, the 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 if there is a dose-dependent response between the amount of lipid ingested and the secretion of the hormones in vivo. Recently, we have found that the concentration of the incretin hormones is higher in intestinal lymph than in peripheral or portal plasma. We therefore used the lymph fistula rat model to test if a dose-dependent trend exists 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, 4.4 kcal). Lymph was continuously collected for 3 h and analyzed for triglyceride (TG), GIP, and GLP-1 content. In response to increasing lipid calories, the secretion of TG, GIP, and GLP-1 into lymph increased dose-dependently. Interestingly, the GLP-1-secreting cells were more responsive than the GIP-secreting cells to changes in intraluminal lipid content. The differing 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.