Lipid, particularly long-chain triglyceride, initiates feedback regulation of gastrointestinal function. To determine whether the site of action of lipid is pre- or postabsorptive, we investigated the ability of mesenteric lipid-fed lymph to inhibit gastric motor function. Lymph was collected from awake lymph-fistula rats during intestinal infusion with either a glucose-saline maintenance solution or lipid. Intra-arterial injection of lymph collected during intestinal lipid infusion significantly inhibited gastric motility in anesthetized recipient rats compared with injection of equivalent amounts of triglyceride or lymph collected during intestinal infusion of maintenance solution. Lymph collected from rats during lipid infusion with Pluronic L-81 [an inhibitor of chylomicron formation and apolipoprotein (apo) A-IV secretion] compared with lymph injection from donor animals treated with Pluronic L-63 (a noninhibitory control for Pluronic L-81) was significantly less potent. Injection of purified recombinant apo A-IV significantly inhibited gastric motility. Products of lipid digestion and absorption, other than fatty acids or triglyceride, released by the intestine during lipid digestion likely serve as signals to initiate intestinal feedback regulation of gastrointestinal function. Most likely, apo A-IV is one of the signals involved.