Discoidal reconstituted high density lipoproteins (rHDL) with a diameter of 7.9 nm, a molar ratio of egg phosphatidylcholine (PC): unesterified cholesterol (UC): cholesteryl esters (CE): apolipoprotein (apo) A-I of 33:7:0:1 and containing two molecules of apoA-1 per particle were incubated with lecithin:cholesterol acyltransferase (LCAT) in the presence of low density lipoproteins as a source of additional UC and PC for the LCAT reaction. After 24 h of incubation, the rHDL had a diameter of 8.8 nm, a molar ratio of PC:UC:CE:apoA-I of 16:3:23:1 and contained three rather than two molecules of apoA-I per particle. The fact that there was no change in the concentration of rHDL-associated apoA-I indicated that the increase from two to three molecules of apoA-I per particle was achieved at the expense of a one-third reduction in the number of rHDL particles in a process that must have involved particle fusion. When the incubations were repeated in the presence of exogenous, lipid-free apoA-I, the resulting rHDL were identical in size and composition to those generated in its absence. Under these conditions, however, the increase from two to three molecules of apoA-I per rHDL particle coincided with a 50% increase in the concentration of rHDL-associated apoA-I. Thus, when lipid-free apoA-I is available, the LCAT-mediated increase in number of apoA-I molecules per rHDL particle is achieved by a direct incorporation of lipid-free apolipoprotein without any need for particle fusion and therefore without a reduction in the number of rHDL particles.