HDLs encompass structurally heterogeneous particles that fulfill specific functions in reverse cholesterol transport. Two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) of normal plasma and subsequent immunoblotting with anti-apolipoprotein (apo) A-I antibodies differentiates an abundant particle with electrophoretic alpha-mobility and less abundant particles with electrophoretic pre-beta-mobility (pre beta 1-LpA-I, pre beta 2-LpA-I, pre beta 3-LpA-I). Immunodetection with anti-apoA-II antibodies identifies a single particle with alpha-mobility. To differentiate alpha-migrating HDL without apo A-II (alpha-LpA-I) from those with apoA-II (alpha-LpA-I/A-II), we combined 2D-PAGGE with immunoadsorption of apoA-II. Incubation of plasma with [3H]cholesterol-labeled fibroblasts in combination with immunosubtracting 2D-PAGGE allowed us to analyze the role of alpha-LpA-I and alpha-LpA-I/A-II in the uptake and esterification of cell-derived cholesterol in native plasma. Depending on the duration of incubations with cells, alpha-LpA-I took up two to four times more [3H]cholesterol than alpha-LpA-I/A-II. Irrespective of the duration of incubation, two to three times more [3H]cholesteryl esters accumulated in alpha-LpA-I than in alpha-LpA-I/A-II. Subsequent incubations in the presence of an inhibitor of lecithin:cholesterol acyltransferase led to preferential accumulation of [3H]cholesteryl esters in alpha-LpA-I/A-II. In conclusion, our data indicate that alpha-LpA-I is more effective than alpha-LpA-I/A-II in both uptake and esterification of cell-derived cholesterol. Moreover, alpha-LpA-I/A-II appears to accumulate cholesteryl esters, at least partially, from alpha-LpA-I.