The effects of cholesteryl ester transfer protein (CETP) on the direct uptake of HDL cholesteryl ester by the liver was investigated using the rat in vivo and the isolated perfused rat liver as experimental models. Rat plasma was incubated with [3H]cholesterol in the presence or absence of partially purified human CETP for 18 hr and [3H] cholesteryl ester-labeled HDL was then isolated by ultracentrifugation. The CETP-treated as compared to untreated HDL showed a small shift toward a lower density in the peak of lipoprotein cholesterol, suggesting that the HDL particle size was increased. After injection of the labeled HDL into rats in vivo, more radioactivity remained in the plasma after 60 min when the CETP-treated preparation was used, but the amounts found in the liver and secreted in the bile were not significantly different from those obtained with the untreated HDL. The distribution of the label remaining in the plasma after 60 min between different density fractions corresponding to HDL subclasses suggested that the uptake of HDL2 and HDL3 was delayed by CETP treatment. Radioactivity from CETP-treated HDL was also removed from the perfusate of isolated perfused rat livers more slowly than that from untreated HDL, and in this case the amount found in the liver after 60 min was significantly lower. These findings indicate that treatment with CETP has a direct inhibitory effect on the clearance of rat HDL cholesteryl ester from the blood and its uptake by the liver.