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This article is available online at http://www.jlr.org Supplementary key words high density lipoprotein • kidney • megalin • chloride-proton exchanger ClC5 • cystinosin Sphingosine-1-phosphate (S1P) acts both as an intracellular signaling molecule and an extracellular agonist of at least fi ve different G protein-coupled receptors. By its dual functions, S1P regulates the survival, proliferation, and migration as well as the functionality of many cells, eventually in opposite directions ( 1-4 ). Therefore the absolute and relative abundance of S1P in intracellular and extracellular compartments appears to be important for its biological functionality ( 4 ).Most cells form S1P by the phosphorylation of sphingosine, a degradation product of ceramides, through sphingosine kinase and degrade S1P to phosphoethanolamine and fatty aldehyde through S1P-lyase ( 1-4 ). By contrast, not only erythrocytes and platelets, but also other cells which have low or no lyase activity, release S1P ( 4-6 ), probably by an as yet unidentifi ed ABC transporter ( 4,7,8 ). Within the plasma compartment, the majority of S1P is transported by HDLs in which it exerts many cyto-protective and anti-infl ammatory effects, for example, on endothelial cells ( 8-10 ). The enrichment of S1P in HDL has been explained by the presence of a specifi c S1P binding protein, namely apoM ( 10 ). Purifi ed and recombinant apoM binds S1P with an IC 50 of 0.9 mol/l, which is in the range of physiological S1P plasma concentrations ( 11 ). X-ray crystallography of apoM identifi ed an S1P binding domain which was confi rmed by the recombinant generation of non-S1P binding apoM mutants ( 11 ). In agreement with these physicochemical data, S1P was copurifi ed with apoM containing HDL, but not apoM-free HDL, from both human Abstract Sphingosine-1-phosphate (S1P) mediates several cytoprotective functions of HDL. apoM acts as a S1P binding protein in HDL. Erythrocytes are the major source of S1P in plasma. After glomerular fi ltration, apoM is endocytosed in the proximal renal tubules. Human or murine HDL elicited time-and dose-dependent S1P effl ux from erythrocytes. Compared with HDL of wild-type (wt) mice, S1P effl ux was enhanced in the presence of HDL from apoM transgenic mice, but not diminished in the presence of HDL from apoM knockout ( Apom ؊ / ؊ ) mice. Artifi cially reconstituted and apoM-free HDL also effectively induced S1P effl ux from erythrocytes. S1P and apoM were not measurable in the urine of wt mice. Apom ؊ / ؊ mice excreted signifi cant amounts of S1P. apoM was detected in the urine of mice with defective tubular endocytosis because of knockout of the LDL receptor-related protein, chloride-proton exchanger ClC-5 ( Clcn5 ؊ / ؊ ), or the cysteine transporter cystinosin. Urinary levels of S1P were signifi cantly elevated in Clcn5 ؊ / ؊ mice. In contrast to Apom ؊ / ؊ mice, these mice showed normal plasma concentrations for apoM and S1P. In conclusion, HDL facilitates S1P effl ux from erythrocytes by both apoMdependent and apoM-independent mechanisms...