Exaggerated postprandial lipemia is associated with coronary heart disease and type II diabetes, yet few studies have examined the effect of sequential meals on lipoprotein metabolism. We have used 13C-labeled fatty acids to trace the incorporation of fatty acid derived from a meal into apolipoprotein B-100 (apoB-100)-containing lipoproteins and plasma nonesterified fatty acids (NEFA) following two consecutive meals. Healthy volunteers (n=8) were given breakfast labeled with [1-(13)C]palmitic acid, eicosapentaenoic acid, and docosahexaenoic acid, followed 5 h later by lunch containing [1-(13)C]oleic acid. Blood samples were taken over a 9-h period. ApoB-100-containing lipoproteins were isolated by immunoaffinity chromatography. Chylomicron-triacylglycerol (TG) concentrations peaked at 195 min following breakfast but at 75 min following lunch (P<0.001). VLDL-TG concentrations, in contrast, rose to a broad peak after breakfast and then fell steadily after lunch. Breakfast markers followed chylomicron-TG concentrations and appeared in plasma NEFA with a similar profile, whereas [1-(13)C]oleic acid peaked 2 h after lunch in plasma TG and NEFA. Breakfast markers appeared steadily in VLDL, peaking 1-3 h after lunch, whereas [1-(13)C]oleic acid was still accumulating in VLDL at 9 h. Around 17% of VLDL-TG originated from recent dietary fat 5 h after breakfast, and around 40% at the end of the experiment. We conclude that there is rapid flux of fatty acids from the diet into endogenous pools. Further study of these processes may open up new targets for intervention to reduce VLDL-TG concentrations and postprandial lipemia.