Plasma high density lipoprotein-3 (HDL3) subfractions with different composition and electric charge properties were isolated by anion exchange chromatography; their ability to exchange cholesteryl esters with low density lipoproteins (LDL) in the presence of the human cholesteryl ester transfer protein (CETP) was studied. The rate of radiolabeled cholesteryl esters transferred between LDL and HDL3 was progressively enhanced as the negative charge density of HDL3 particles increased, until the maximal transfer value was reached with a charge density ranging between -2,200 and -2,250 esu/cm2. Consistent data were obtained when cholesteryl ester transfer was measured either from radiolabeled LDL towards HDL3 or from radiolabeled HDL3 towards LDL. In both cases, a progressive decrease in the cholesteryl ester transfer rate was observed as the charge density increased above the optimal value. When HDL3 particles were progressively enriched with apoA-II with no modification of their lipid content, the electronegative charge progressively decreased. In good agreement with data obtained with native HDL3 subfractions isolated from human plasma, the rate of radiolabeled cholesteryl esters transferred from LDL towards apoA-II-enriched HDL3 increased progressively as the density of negative charge increased, until an optimal surface charge density of approx. -2,200 esu/cm2, was reached. As the charge density of apoA-II-enriched HDL3 exceeded the optimal value, the cholesteryl ester transfer rate was substantially reduced. Consistent observations were made by substituting apoA-II for apoA-I either in immunopurified HDL3 particles containing mainly apoA-I or in the plasma HDL3 subfractions with the highest electronegativity. It is concluded that the charge density of plasma HDL3 constitutes one major determinant of maximal CETP activity.