Reverse cholesterol transport (RCT) is a complex process. Several steps, utilizing enzymes and transfer proteins, occur within the plasma compartment before the cholesterol molecules are delivered from peripheral cells to hepatocytes (Atherosclerosis 88:99-107, 1991; Arterioscler Thromb Vasc Biol 21:13-27, 2001). Both intracellular and extracellular factors affect the direction of cholesterol flux between cells and serum. When cells are incubated with serum in vitro there is flux of free cholesterol (FC) out of the cell (efflux) while simultaneously there is movement of lipoprotein FC and cholesteryl ester (CE) into the cell (influx). Net cholesterol flux is the difference between influx and efflux and this difference may result in net accumulation, net depletion, or no change in cell cholesterol content. The protocols to measure the efflux of labeled cholesterol are relatively simple since only FC undergoes efflux and the assay can be easily adapted for high throughput use. A significant relationship between net cholesterol removal and the fractional efflux of tracer cholesterol is seen in cholesterol-enriched cells indicates that in vitro isotopic measures of serum efflux efficiency may be valid surrogate measure of the capacity of human serum to remove excess tissue cholesterol in vivo. We have also measured net cholesterol flux as the change in cell cholesterol content upon incubation of J774 cells and mouse peritoneal macrophages with human serum. We found that the cholesterol content of the cell modulates the relative contribution of efflux pathways (ABCAI, SRBI, ABCGI) and thus changes the cell’s response to HDL subfractions and the direction of net cholesterol flux.