The in vivo incorporation of labeled amino acids into two forms of apolipoprotein B of nascent hepatic, nascent intestinal, and plasma lipoproteins was studied. Using SDS-gel filtration column chromatography rat apolipoprotein B was separated into two proteins of higher (apo Bh) and of lower (apo Bl) molecular size and the incorporation of label into each was measured. When livers isolated from fed rats were perfused with 3H-labeled amino acids, radioactivity was incorporated into both forms of apo B of the d less than 1.060 fractions (very low (VLDL), intermediate (IDL), and low (LDL) density lipoproteins) with a labeling ratio of apo Bl to apo Bh of 0.8. When mesenteric lymph was collected from corn oil fed rats intraduodenally injected with 3H-labeled amino acids, radioactivity was mainly incorporated into apo Bl of chylomicrons and VLDL with apo Bl to apo Bh labeling ratios of 14 and 44, respectively. Plasma was isolated 2 h after intraperitoneal injection of 3H-labeled amino acids into chow fed rats and lipoproteins were isolated by sequential density ultracentrifugation. The labeling ratio of apo Bl and apo Bh decreased from 4.2 in VLDL to 0.5 in LDL indicating a progressive enrichment of apo Bh in the LDL fraction. High density lipoproteins (HDL) contained less than 4% of the total labeled apo B and was enriched in apo Bl. The results of this study indicate that the liver synthesizes both forms of apo B while the intestine synthesizes almost entirely apo Bl. Since both apo B proteins are secreted primarily by the liver into VLDL, the results are consistent with preferential removal of apo Bl during triglyceride-rich lipoprotein catabolism and entry of hepatically derived apo Bh into LDL.