Binding studies at 37 °C showed that lipoprotein lipase-treated very low density lipoproteins (LPL-VLDL) and very low density lipoproteins (VLDL), once taken up via the low density lipoprotein (LDL) receptor, are poorly degraded by HepG2 cells as compared with LDL. Determination of the initial endocytotic rate for LPL-VLDL and VLDL as compared to LDL shows that LPL- VLDL and VLDL are internalized at a similar rate as LDL. Incubation of cells with labeled LDL, LPL-VLDL, and VLDL at 18 °C for 4.5 h resulted in the accumulation of these particles in the early endosomes, without subsequent transport to the lysosomes and degradation. After washing the cells and a temperature shift to 37 °C, the labeled LDL present in the early endosomes is transported to the lysosomal compartment almost completely within 15 min. Strikingly, for LPL-VLDL and for VLDL, only about 50% or less of the label was moved to the lysosomal compartment within 45 min. However, once present in the lysosomes, VLDL and LPL-VLDL are degraded about 1.6-fold more rapidly than LDL. Retroendocytosis accounts for less than 10% of the internalized LDL, whereas a higher rate of retroendocytosis, up to 20 and 40%, respectively, was observed for LPL-VLDL and VLDL. To evaluate the effect of the inefficient transport of VLDL and LPL-VLDL to the lysosomal compartment on cellular cholesterol homeostasis, acyl-CoA:cholesterol acyltransferase (ACAT) activity was measured. Incubation with 30 ug/ml of LDL induced a 2.5- fold increase in ACAT activity, whereas the incubation with similar amounts of both VLDL and LPL-VLDL failed to stimulate this enzyme. We conclude that both a slower transport to the lysosomal compartment and a higher rate of retroendocytosis, possibly as the consequence of the longer residence time in the early endosomes, are responsible for the poor degradation of VLDL and LPL-VLDL by HepG2 cells. Chemicals/CAS: cholesterol acyltransferase, 9027-63-8; Cholesterol, 57-88-5; Lipoprotein Lipase, EC 3.1.1.34; Lipoproteins, LDL; Lipoproteins, VLDL; Sterol O-Acyltransferase, EC 2.3.1.26; Triglycerides