The cholesteryl ester flow between high-density lipoproteins (HDL) and triglyceride-rich lipoproteins was investigated utilizing HDL fractions and synthetic lipid emulsion particles (EM). HDL was labeled in vitro with [1,2-3H(n)]cholesteryl ester or with [1,2,6,7-3H(n)]cholesteryl oleate, whereas EM were made with [4-14C]cholesteryl oleate (CO) or [carboxyl-14C]triolein (TO). The cholesteryl ester (CE) transfer rate between HDL and EM was spontaneous to some extent (because it occurred in saline medium), saturable, enhanced in a dose-dependent manner by the plasma fraction at D > 1.21 g/ml ascribed to its CETP activity, and greater for HDL3 than for HDL2. Unesterified fatty acids in the medium elicited two opposing effects: (1) enhanced the spontaneous cholesteryl oleate shift to EM and inhibited the reverse flow from EM to HDL3 but not to HDL2; (2) partially impaired the plasma D > 1.21 g/ml-induced bidirectional cholesteryl oleate flow. Approx. 2 mol TO from EM exchange for 1 mol CO from HDL3. Net cholesteryl ester balance was dependent on the concentration of HDL-cholesteryl ester, and independent from EM-cholesteryl oleate, possibly due to the much smaller concentration of the latter. These in vitro experiments shed light on the complex physical chemistry of transport of cholesteryl ester and triolein between HDL and TG-rich lipoproteins which occurs during the metabolism of chylomicrons in plasma.