Abstract-Cholesterol efflux from macrophage foam cells is a rate-limiting step in reverse cholesterol transport. In this process cholesterol acceptors like high-density lipoproteins (HDL) and apolipoprotein (apo)A-I must cross the endothelium to get access to the donor cells in the arterial intima. Previously, we have shown that apoA-I passes a monolayer of aortic endothelial cells (ECs) from the apical to the basolateral side by transcytosis, which is modulated by the ATP-binding cassette transporter (ABC)A1. Here, we analyzed the interaction of mature HDL with ECs. ECs bind HDL in a specific and saturable manner. Both cell surface biotinylation experiments and immunofluorescence microscopy of HDL recovered Ϸ30% of the cell-associated HDL intracellularly. Cultivated on inserts ECs bind, internalize, and translocate HDL from the apical to the basolateral compartment in a specific and temperature-dependent manner. The size of the translocated particle was reduced, but its protein moiety remained intact. Using RNA interference, we investigated the impact of SR-BI, ABCA1, and ABCG1 on binding, internalization, and transcytosis of HDL by ECs. HDL binding was reduced by 50% and 30% after silencing of SR-BI and ABCG1, respectively, but not at all after diminishing ABCA1 expression. Knock down of SR-BI and, even more so, ABCG1 reduced HDL transcytosis but did not affect inulin permeability. Cosilencing of both proteins did not further reduce HDL binding, internalization, or transport. In conclusion, ECs transcytose HDL by mechanisms that involve either SR-BI or ABCG1 but not ABCA1. cholesterol show an inverse association with the incidence of coronary artery disease. The cardioprotective effect of HDL and its major apolipoprotein (apo)A-I are, in part, related to the ability to promote the reverse transport of cholesterol from macrophage foam cells in the arterial intima to the liver for excretion into the bile. [1][2][3][4] An early step in the reverse transport of cholesterol is the transfer of excess cholesterol from the lipid-laden macrophages to HDLs. Importantly, the loading of cellular cholesterol to HDL does not take place in the plasma compartment but in the subendothelial space of arteries. 1 Consequently, HDLs must cross the endothelium to get into close proximity to the cholesterol donor cells. This passage is not well understood. 5 The endothelium lines the vasculature as a single layer of endothelial cells (ECs). As a semipermeable barrier, it regulates the flux of liquid, solutes, and cells between blood and interstitial space. Two principal pathways are known for transendothelial macromolecule translocation, the transcellular transport, including transcytosis, and the paracellular transfer between adjacent cells. 6,7 The paracellular pathway is formed by gaps between ECs, but regulated adherence and tight junctions restrict and control the free passage of macromolecules larger than 6 nm. 7 Endothelial transcytosis, which is defined as vesicle-mediated transport of proteins, has been best investigated ...