Peripheral cholesterol can be transported back to liver by acceptors, particularly HDL, for disposal. The properties of cholesterol acceptors have been studied extensively. It has been demonstrated that the ability of serum to promote cholesterol efflux from Fu5AH rat hepatoma cells correlated best with the phospholipid content of HDL. In our studies, serum was enriched with phospholipid and the consequences of phospholipid enrichment were studied. We found that the ability of serum to elicit efflux of cholesterol could be enhanced by phospholipid modification in Fu5AH cells, suggesting phospholipid is a key parameter in modulating cellular cholesterol efflux. To elucidate the mechanism(s) by which PL-modification of serum influences efflux, we have proposed several possibilities: (1) lipoprotein surface area is increased resulting in increased cholesterol solubilization; (2) new HDL-like particles are generated, increasing the numbers of cholesterol acceptors; and (3) PL alters the surface structure of lipoproteins, particularly HDL, increasing their affinity for a receptor on the cell surface, such as SR-BI, the HDL receptor which mediates cholesterol ester uptake. To discriminate among these alternatives, cholesterol efflux studies were performed in a variety of cell lines which express different levels of SR-BI. Our results show that the rate of cholesterol release from different cell types varies significantly and that phospholipid-modification of serum can enhance the efflux ability of serum in some cell types but not others. Further studies demonstrated that the percentage of efflux stimulation by phospholipid-modification of serum is correlated to the expression levels of SR-BI in cells. In addition, a strong correlation between efflux to 1-palmitoyl-2-oleoyl phosphotidylcholine small unilamellar vesicles (POPC-SUV) and levels of SR-BI was observed, suggesting that neutral phospholipid vesicles can also interact with SR-BI. We conclude that phospholipids can modulate the interactions of lipoprotein with SR-BI and that enrichment of serum, particularly high density lipoprotein (HDL) particles, with phospholipid can increase their ability to bind to SR-BI and thus increase their efflux ability.