Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL apolipoproteins occurs by a non-endocytotic pathway that results in net delivery of cholesteryl esters to cells. With respect to the cellular mechanism of this pathway, previous studies with adrenal cells showed a cholesteryl ester pool that is reversibly associated with cells and which appears to mediate irreversible selective uptake. A cholesteryl ester pool with similar properties was observed in plasma membranes isolated from adrenal cells, suggesting that this is the site of the cellular pool. Human Hep G2 hepatoma cells also selectively take up HDL cholesteryl esters. Therefore we asked if these cells have a reversibly cell-associated cholesteryl ester pool as well that could mediate irreversible selective uptake. To do this, human HDL3 (d = 1.125-1.21 g/ml) was labeled in both its protein and cholesteryl ester moieties. Uptake of HDL3 tracers by Hep G2 cells was then studied. After an uptake incubation in the presence of labeled HDL3, either cellular uptake of tracers was immediately determined or cells were 'chase' incubated in the presence of unlabeled HDL before determination of cellular tracer content. Hep G2 cells selectively took up HDL3 cholesteryl esters under these conditions. However, a fraction of cholesteryl ester tracer selectively taken up was chased from the cells by subsequent incubation in the presence of unlabeled HDL. This reversible pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL3 particles bound to the cell surface. Selective uptake was down-regulated by prior incubation with LDL, and cholesteryl ester tracer in the reversible pool was down-regulated in parallel. Plasma membranes were isolated from Hep G2 cells and incubated with doubly labeled HDL3. HDL3 particles bound to these membranes, as indicated by the apolipoprotein tracer. However, HDL cholesteryl esters associated with plasma membranes in excess on that accounted for by HDL3 particles. This selective association of HDL3 cholesteryl ester tracer with membranes was reversible, and the tracer was chased during incubation in the presence of unlabeled HDL. These results suggest that, as with steroidogenic cells, a reversible pool of cholesteryl esters localized in the plasma membrane is involved in selective uptake of HDL3 cholesteryl esters by hepatic cells at a step prior to irreversible internalization.