A protein band having the same migration as apolipoprotein (apo) B-48 was observed by SDS electrophoresis in the plasma very low density lipoprotein (VLDL) from 14 Type IV and three Type III hyperlipoproteinemic subjects and from six normal fasting subjects. The VLDL from five Type IV, three Type III, and one normal subject were separated into two subfractions, retained and nonretained, by immunoaffinity chromatography on monoclonal anti-apo B-100 Sepharose. Based on results of electrophoresis and radioimmunoassay, we have concluded that these two fractions represent apo B-48 and apo B-100 lipoproteins that we have named apo B-48 and apo B-100 VLDL. When compared to their respective apo B-100 VLDL, the apo B-48 VLDL from either Type III or Type IV was principally enriched in total lipids, in apo E, and had an electrophoretic migration similar to chylomicrons. This suggests that apo B-48 VLDL has the same origin (i.e., intestinal) in the two disorders. Both apo B-48 and apo B-100 VLDL were enriched in cholesteryl ester (CE) and depleted in triglyceride (TG) in Type III; however, both fractions were rich in TG and poor in CE in Type IV and in normal subjects. In addition, compared to their respective apo B-100 VLDL, the apo B-48 fraction was enriched in CE in Type III and in TG in Type IV. We conclude that, despite a possible similar origin for apo B-48 VLDL in Type III and in Type IV subjects, the composition of apo B-48 VLDL is variable and the CE/TG ratio is more characteristic of the type of hyperlipidemia than of the particular VLDL subfractions.