Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 6 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression. Although all cells synthesize cholesterol, only liver and steroidogenic tissues effect net cholesterol removal. Reverse cholesterol transport (RCT) from peripheral tissues to liver for oxidation and excretion is the major physiological pathway for the net cholesterol elimination from the body. Although increasing evidence indicates that plasma membrane microdomains, termed lipid rafts/caveolae, compartmentalize cell surface proteins that mediate HDLcholesterol uptake/efflux in peripheral cells, the existence and/or significance of such domains in hepatocyte plasma membranes is not completely clear, especially in view of the nearly negligible levels of caveolin-1 in liver. However, an overall scheme is beginning to evolve based largely on findings with peripheral cells (reviewed in Ref. 1).First, peripheral cells (fibroblasts, muscle, and endothelial cells) are rich in both lipid rafts and caveolae (a subset of lipid rafts rich in several components of the RCT pathway) but are relatively poor in intracellular cholesterol binding proteins such as sterol carrier protein-2 (SCP-2) that facilitate cholesterol retention rather than efflux (reviewed in Refs. 1, 2). Cholesterol effluxes from peripheral cells to HDL by a process facilitated by: i) the HDL receptor scavenger receptor class B type I (SR-B1) (3); ii) ABCA1, a protein that mediates cholesterol and phosphatidylcholine desorption from the pla...