The nature of the interaction of high density lipoproteins (HDL), formed during lipolysis of human very low density lipoprotein (VLDL) by perfused rat heart, with subfractions of human plasma HDL was investigated. Perfusate HDL, containing apoliproproteins (apo) E, C-II, and C-III but no apo A-I or A-II, was incubated with a subfraction of HDL (HDL-A) containing apo A-I and A-II, but devoid of apo C-II, C-III, and E. The products of the incubation were resolved by heparin-Sepharose or hydroxylapatite chromatography under conditions which allowed the resolution of the initial HDL-A and perfusate HDL. The fractions were analyzed for apolipoprotein content and lipid composition and assessed for particle size by electron microscopy. Following the incubation, the apo-E-containing lipoproteins were distinct from perfusate HDL since they contained apo A-I as a major component and apo C-II and C-III in reduced proportions. However, the HDL-A fraction contained apo C-II and C-III as major constituents. Associated with these changes in apolipoprotein composition, the apo-E-rich lipoproteins acquired cholesteryl ester from the HDL-A fraction and lost phospholipid to the HDL-A fraction. The HDL-A fraction maintained a low unesterified cholesterol/phospholipid molar ratio (0.23), while the apo-E-containing lipoproteins possessed a high ratio (0.75) characteristic of the perfusate HDL. The particle size of apo-E-containing lipoproteins (138.9 ± 22.5 Å; 1 Å = 0.1 nm) was larger than the initial HDL-A (126.5 ± 17.6 Å) or the new HDL-A-like fraction (120.9 ± 17.4 Å) obtained following incubation with perfusate HDL. It is concluded that incubation of perfusate HDL containing apo E, C-II, and C-III with plasma HDL subfractions results in the acquisition of apo A-I and cholesteryl esters by the apo-E-containing perfusate HDL and the loss of apo C-II, C-III, and phospholipid to the plasma HDL-A fraction. The process does not appear to be due to fusion of the particles, since the apo-E-containing lipoproteins maintain a cholesterol/phospholipid ratio distinct from the HDL-A fraction. The data provide evidence for a potential mechanism for the formation of HDL-E, an apo-E-containing lipoprotein of HDL size and density, through lipolysis of VLDL.