Cholesteryl ester-rich beta-very low density lipoproteins (beta-VLDL) are beta-migrating lipoproteins that accumulate in the d < 1.006 g/ml fraction of plasma from cholesterol-fed animals and from patients with Type III hyperlipoproteinemia. They can be separated from pre-beta-migrating very low density lipoproteins in the d 1.006 g/ml fraction by Geon-Pevikon block electrophoresis. The beta-VLDL have a general property of stimulating cholesteryl ester synthesis and accumulation in macrophages. In the present study, we demonstrated that beta-VLDL obtained from cholesterol-fed dogs fasted for 16 hr were heterogeneous and that two subpopulations of particles, referred to as Fractions I and II, could be isolated from the whole beta-VLDL fraction using gel filtration chromatography. These fractions of beta-VLDL were similar in that both were cholesteryl ester rich, had beta-electrophoretic mobility on Geon-Pevikon electrophoresis, and possessed the B and E apoproteins as major constituents. However, Fractions I and II differed in size, shape, electrophoretic mobility, chemical composition, and apoprotein B type. (Fraction I vs. Fraction II: size: 90 to 300 nm vs. 20 to 70 nm; shape: irregular with redundant surface vs. spherical; electrophoretic mobility on paper: origin vs. beta; chemical composition: rich in phospholipid and poor in protein vs. rich in protein and poor in triglycerides; apoprotein B types: equal amounts of the high and low molecular weight forms vs. predominantly the high molecular weight form.) Furthermore, Fraction I was 3- to 15-fold more active than Fraction II in stimulating cholesteryl ester formation in mouse peritoneal macrophages. The concentration of Fraction I, but not Fraction II, was diminished in plasma by prolonged fasting, and Fraction I transported more intestinal-absorbed retinol than Fraction II. In addition, the plasma clearance of Fraction I injected into cholesterol-fed dogs was distinctly different from the clearance of Fraction II, and the in vivo dieaway of Fraction I resembled that of chylomicrons and chylomicron remnants. These findings suggest that beta-VLDL in dogs are composed of cholesteryl ester-rich chylomicron remnants (Fraction I) and cholesteryl ester-rich lipoproteins, probably of liver origin (Fraction II). Finally, in studies of two patients with Type III hyperlipoproteinemia, we also identified the existence of two fractions in the beta-VLDL with characteristics similar to Fractions I and II of cholesterol-fed dogs.-Fainaru, M., R. W. Mahley, R. L. Hamilton, and T. L. Innerarity. Structural and metabolic heterogeneity of beta-very low density lipoproteins from cholesterol-fed dogs and from humans with Type III hyperlipoproteinemia.