1. High density lipoprotein (HDL) of the New Zealand White rabbit was separated by heparin-Sepharose affinity chromatography into six distinct subfractions of different composition and particle size. 2. When human acetyl LDL containing [3H]cholesteryl linoleate was injected intravenously into rabbits to prime the endothelial cells with labelled cholesterol, only 1-2% of the radioactivity remained in the plasma after 2 hr. 3. After 4 hr, 60.1% of the plasma radioactivity was present in HDL and 25% of this was recovered in the largest particles of HDL (fraction VI, mean particle diameter 11.6-11.8 nm). 4. The concentration of these largest particles of HDL, rich in apolipoprotein E, were also relatively increased in acetyl-LDL-treated rabbits when compared to controls (P < 0.01). 5. In control in vitro experiments, 62.2% of the radioactivity recovered in HDL was associated with subfractions IV and V (mean particle diameter 10.2-10.8 nm) while only 5% was present in fraction VI. 6. The results show that large HDL particles enriched with apo E contain a large proportion of cholesterol previously supplied to hepatic and peripheral endothelial cells. 7. This study demonstrated that the rabbit provides a useful animal model for the study of the metabolism of subfractions of HDL in relation to reverse cholesterol transport.