Saposin A (Sap-A) is one of five known sphingolipid activator proteins (SAPs) required for the lysosomal degradation of sphingolipids and for the loading of lipid antigens onto antigen presenting molecules of the CD1 type. Sap-A assists in the degradation of galactosylceramide by galactosylceramide-ß-galactosidase in vivo, that takes place at the surface of intraendosomal/intralysosomal vesicles. Sap-A is believed to mediate the interaction between the enzyme and its membrane-bound substrate. Its dysfunction causes a variant form of Krabbe disease. In the present study we prepared glycosylated Sap-A free of other Saps taking advantage of the P. pastoris expression system. Using liposomes and surface plasmon resonance (SPR) spectroscopy, we tested the binding and lipid mobilization capacity of Sap-A under different conditions. Along the endocytic pathway, the pH-value decreases and the lipid composition of intraendosomal and intralysosomal membranes changes drastically. In the inner membranes, the cholesterol concentration decreases, and that of the anionic phospholipid bis(monoacylglycero)phosphate (BMP) increases. Here, we show that Sap-A is able to bind to liposomes and to mobilize lipids out of them at acidic pH values below pH 4.7. Low cholesterol levels and increasing concentrations of BMP favor lipid extraction significantly. Galactosylceramide as bilayer component is not essential for lipid mobilization by Sap-A, that requires intact disulfide bridges for activity. We also show for the first time that glycosylation of Sap-A is essential for its lipid extraction activity. Variant Sap-A proteins, which cause storage of galactosylceramide in humans (Krabbe disease, Spiegel, et al., (2005) Mol Genet Metab 84, 160-166.) and in mutant mice (Matsuda, J. el al., (2001) Hum Mol Genet 10, 1191-1199) are deficient in lipid extraction capacity.