Pulse-chase incubations of human plasma with [3H]cholesterol-laden skin fibroblasts or low density lipoproteins (LDL) and nondenaturing two-dimensional electrophoresis were used to study the transfer and esterification of cell-derived unesterified cholesterol (UC) in human plasma lipoproteins. Specific radioactivities ([3H]UC per microgram of UC) were calculated, and net cholesterol mass transfer was quantified using a fluoro-enzymatic assay to validate productive transfers of UC between high density lipoprotein (HDL) and LDL. Cellular UC was initially taken up by pre-beta 1-HDL and subsequently transferred in the sequence pre-beta 2-HDL-->pre-beta 3-HDL-->alpha-HDL-->LDL. During the first 5 minutes of this process, only 5% of cellular cholesterol was esterified in pre-beta 3-HDL and alpha-HDL; the remainder reached LDL as UC. Cellular UC accumulating in LDL was then redistributed to various HDL particles via two pathways: 1) the partially LDL receptor-mediated uptake and re-secretion of UC by cells and 2) the direct transfer of UC to HDL, mostly to alpha-HDL and a small amount to pre-beta-HDL. UC was not transferred from LDL to HDL after inhibition of lecithin:cholesterol acyltransferase (LCAT). The esterification of cellular [3H]cholesterol in plasma was competitively inhibited by the addition of excess unlabeled LDL but not of excess HDL. However, both excess LDL and excess HDL prevented the esterification of cell-derived cholesterol in apolipoprotein B-free plasma. This demonstrated that LDL is the major source of UC to the LCAT reaction and that the transfer of UC from LDL to HDL is LCAT dependent. In conclusion, the effective esterification of cell-derived cholesterol in plasma involves a rapid transfer of UC via HDL particles to LDL, from which it is distributed to pre-beta-HDL and alpha-HDL. Furthermore, we hypothesize that the transfer per se of cellular UC to LDL forms a cholesterol concentration gradient between cell membranes and HDL and thus a second, reverse cholesterol transport mechanism in addition to the esterification of cholesterol by LCAT.