To evaluate the hypothesis that stimulation of intestinal apolipoprotein (apo) A-IV by dietary fat depends upon assembly and transport of chylomicrons, we examined the effect of duodenal infusion of fatty acids of graded chain length on mucosal synthesis and lymphatic output of lipid and apo A-IV. Rats with duodenal cannulas and mesenteric lymph fistulas were given 8-h duodenal infusions of lipid emulsions containing either butyric (4:0), caprylic (8:0), lauric (12:0), myristic (14:0), stearic (18:0), oleic (18:1), linoleic (18:2) or arachidonic (20:4) acids, or tributyrin, tricaprylin or triolein. Lymph outputs of triglyceride, phospholipid and apo A-IV were measured at 0, 2, 4, 5, 6, 7 and 8 h after the start of lipid infusion. Significant increases in lymph lipid (triglyceride, phospholipid) and apo A-IV output were observed in response to long-chain fatty acids (14:0, 18:0, 18:1, 18:2, 20:4) or triolein; short- or medium-chain fatty acids (4:0, 8:0, 12:0) or tributyrin or tricaprylin produced no significant increase in lymph lipid output above basal levels. Similarly, increased jejunal mucosal synthesis of apo A-IV was observed in response to duodenal infusion of oleic acid but not butyric or caprylic acid. These results provide direct support for the hypothesis that stimulation of apo A-IV by dietary fat depends upon transport of absorbed lipid via chylomicrons in lymph.