Plasma lipoproteins from male rats were isolated by rate zonal centrifugation. Four lipoproteins were identified: VLDL, LDL, HDL1, and HDL2. LDL, HLD1, and some HDL2 distributed within the salt density interval of 1.006-1.085 g/ml, while HDL2 was found in the 1.063-1.21 g/ml interval. HDL3 was not identified in the rat. Rat VLDL is poor in cholesteryl esters (1.5-3.0% of total mass) and nearly lacks the smaller and denser particle subpopulation which is predominant in humans. Rat LDL, containing a relatively large amount of triglyceride (20.2% of total mass) and a small amount of cholesteryl ester (27.5%), could be isolated free of apoproteins other than apoB. HDL1 is a cholesteryl ester-rich lipoprotein that occupies a density interval overlapping both LDL and HDL2. ApoE is the major protein constituent of HDL1; apoA-I, A-IV, and C are also present. Apo-A-I-rich HDL2 is the only human-like HDL subpopulation found in rats. Lipoproteins from fasted and non-fasted rats were essentially similar. Arachidonic acid contributed 56.7% and 72.3% of total cholesteryl ester fatty acids in HDL1 and HDL2, respectively, but only 7.9% and 27.3% in VLDL and LDL, respectively. Palmitic, palmitoleic, and oleic acids were the major cholesteryl ester fatty acids in VLDL and LDL. In vitro incubation of biosynthetically labeled HDL2 cholesteryl ester with rat plasma demonstrated minimal transfer of the labeled cholesteryl ester to VLDL and LDL. These results indicate biological immiscibility of HDL cholesteryl esters with those of lower density lipoproteins. The finding of cholesteryl ester-poor VLDL and LDL and the presence of HDL as larger and less dense subpopulations is compatible with the absence of cholesteryl ester transfer activity in an animal with pronounced LCAT activity.