Two major species of human apolipoprotein (apo) B have been identified, apo B-48 and apo B-100, which are the predominant forms in chylomicrons and very low density lipoproteins (VLDL), respectively. Due to defective hepatic clearance, apo B-48 containing lipoproteins accumulate in the plasma of subjects with type III hyperlipoproteinemia. In the present study, we have used immunoaffinity chromatography to separate type III VLDL into a nonretained (apo B-48 VLDL) and a retained (apo B-100 VLDL) fraction. To achieve complete separation, as determined by electrophoresis and radioimmunoassay, it was necessary to employ two different insolubilized anti-apo B-100 monoclonal antibodies because of immunochemical heterogeneity within the apo B-100 VLDL fraction. The ability to separate apo B-100 VLDL from apo B-48 VLDL shows that the two apo B species are found on different particles. The apo B-48 VLDL had an electrophoretic mobility similar to chylomicrons, whereas the apo B-100 VLDL migrated similarly to total type III VLDL. Both fractions showed a concentration of particles with diameters approximately 100 nm, with apo B-48 VLDL being somewhat more heterogeneous in particle size. The two fractions were qualitatively similar in apolipoprotein composition but apo B-48 VLDL was enriched in apo E, relative to apo B-100 VLDL. Apo B-48 VLDL was enriched in cholesterol esters and deficient in triglycerides and phospholipids when compared with apo B-100 VLDL. The existence of immunochemical heterogeneity in the apo B-100 VLDL may reflect different functional subpopulations of particles within this fraction.