|Abstract:||High density lipoproteins (HDL), which are found as spherical particles in the circulation, contain a core of neutral lipids, mainly cholesterol esters and an outer shell of apolipoproteins, phospholipids, and cholesterol. Discoidal HDL, discovered in the plasma of patients deficient in lecithin cholesterol acyltransferase (LCAT), become spherical in morphology upon incubation with LCAT and an exogenous source of cholesterol due to the accumulation of cholesterol esters in a neutral lipid core. To date, much has been learned about the structure and function of discoidal HDL through the study of homogeneous reconstituted HDL (rHDL) but not spherical HDL. In this work, spherical rHDL are prepared by incubation of discoidal rHDL, containing apo-lipoprotein A-I (apoA-I), cholesterol, and either palmitoyloleoyl-phosphatidylcholine or dipalmitoylphosphatidylcholine, with LCAT and low density lipoproteins. These spherical rHDL are characterized in terms of their composition, size, apoA-I structure and stability, lipid dynamic properties, and reactivity with LCAT, and are compared to discoidal rHDL, isolated native HDL subfractions, and rHDL intermediates in the disc to sphere conversion. The methods used include gradient gel electrophoresis, fluorescence and circular dichroism spectroscopy, and monoclonal antibody binding studies. The structure of apoA-I on the spherical rHDL does not appear to differ greatly from the apoA-I structure on discoidal rHDL, the native HDL subfractions, and the intermediates in the disc to sphere conversion. However, guanidine hydrochloride denaturation experiments indicate the presence of an N-terminal domain in apoA-I which is more stable in the spherical rHDL and a C-terminal domain containing amphipathic $\alpha$-helices which are more stable in the discoidal rHDL. The lipid dynamic properties vary with the lipid composition and the lipid to protein ratios, and the LCAT reactivity is low with the spherical HDL and the intermediates, possibly due to product inhibition. These studies show that a spherical rHDL can be prepared having a similar size, composition, and apoA-I structure to a native HDL subfraction, thus indicating that spherical rHDL are good models for mature, native HDL. Finally, the study of the rHDL intermediates leads to a hypothetical mechanism for the disc to sphere conversion and suggests a possible source of lipid-free apoA-I in the circulation.