The class B type I scavenger receptor, (SR-BI), is a member of the CD36 superfamily of proteins and is a physiologically relevant, high affinity cell surface high density lipoprotein (HDL) receptor that mediates selective lipid uptake. The mechanism of selective lipid uptake is fundamentally different from that of classic receptor-mediated uptake via coated pits and vesicles (e.g. the low density lipoprotein receptor pathway) in that it involves efficient transfer of the lipids, but not the outer shell proteins, from HDL to cells. The abilities of SR-BI and CD36, both of which are class B scavenger receptors, to bind HDL and mediate cellular uptake of HDL-associated lipid when transiently expressed in COS cells were examined. For these experiments, the binding of HDL to cells was assessed using either 125I- or Alexa (a fluorescent dye)-HDL in which the apolipoproteins on the surface of the HDL particles were covalently modified. Lipid transfer was measured using HDL noncovalently labeled by the fluorescent lipid 1,1'-dioctadecyl-3,3, 3',3'-tetramethylindocarbocyanine perchlorate. Although both mSR-BI and human CD36 (hCD36) could mediate the binding of HDL in a punctate pattern across the surfaces of cells, only mSR-BI efficiently mediated the transfer of lipid to the cells. Analysis of point mutants established that the major sites of fatty acylation of mSR-BI are Cys462 and Cys470 and that fatty acylation is not required for receptor clustering, HDL binding, or efficient lipid transfer. Generation of mSR-BI/hCD36 domain swap chimeras showed that the differences in lipid uptake activities between mSR-BI and hCD36 were not due to differences between their two sets of transmembrane and cytoplasmic domains but rather result from differences in their large extracellular loop domains. These results show that high affinity binding to a cell surface receptor is not sufficient to ensure efficient cellular lipid uptake from HDL. Thus, SR-BI-mediated binding combined with SR-BI-dependent facilitated transfer of lipid from the HDL particle to the cell appears to be the most likely mechanism for the bulk of the selective uptake of cholesteryl esters from HDL to the liver and steroidogenic tissues.