The distribution, chemical, and apoprotein composition of plasma and peripheral lymph lipoproteins were compared in control and cholesterol-fed dogs. In both groups of animals, the agarose electrophoretic patterns of plasma and lymph lipoproteins were similar. In hypercholesterolemic dogs, beta-very low density lipoprotein, beta-migrating intermediate density lipoprotein, and HDLc were major components both in plasma and lymph, providing evidence for a potential interaction of these atherogenic particles with macrophages and other peripheral cells. The chemical composition and physical appearance of peripheral lymph HDL was markedly different from that of plasma HDL (high density lipoprotein), especially in the cholesterol-fed animals. Lymph HDL had a higher cholesterol to protein ratio and a markedly increased free cholesterol content (free cholesterol to cholesteryl ester ratio of 1.7 as opposed to 0.2 in plasma HDL in cholesterol-fed animals). The phospholipid content of lymph HDL was higher than that of plasma HDL, while the protein content was lower. A significant proportion of lymph HDL obtained from cholesterol-fed dogs was in the form of disc-shaped particles stacked in rouleau structures. Changes in plasma apolipoprotein concentrations due to cholesterol feeding were reflected in peripheral lymph to different degrees, depending largely on the relative size of the lipoproteins containing the individual lipoproteins. A considerable enrichment of lymph HDL with apoE and apoA-IV was observed by both immunochemical and electrophoretic methods. In lymph HDL from control and cholesterol-fed dogs, the apoE/apoA-I and apoA-IV/apoA-I ratios were several-fold elevated, compared to those of plasma HDL. It is concluded, therefore, that during cholesterol feeding a substantial portion of interstitial HDL is assembled de novo in the periphery as a crucial stage of reverse cholesterol transport to the liver. It is likely that further modification occurs upon entry to plasma and exposure to lecithin:cholesterol acyltransferase, possibly leading to generation of HDLc. Alternatively, these particles may be directly and rapidly removed by the liver.