The specific recognition of anionic phospholipids in the outer leaflets of cell membranes and lipoproteins by cell surface receptors may play an important role in a variety of physiologic and pathophysiologic processes (e.g. recognition of damaged or senescent cells by the reticuloendothelial system or lipoprotein homeostasis). Several investigators have described anionic phospholipid binding to cells, and phosphatidylserine (PS) binding to a partially purified approximately 95-kDa membrane protein has recently been reported (Sambrano, G.R., and Steinberg, D. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 1396-1400). Using both direct binding and ligand competition assays in transfected cells, we have found that two class B scavenger receptors, SR-BI and CD36, can tightly bind PS and phosphatidylinositol (PI)-containing liposomes (Kd for PS liposome binding to SR-BI is approximately 15 micrograms phospholipid/ml or 0.18 nM (mol PS liposomes/l), but not phosphatidylcholine, phosphatidylethanolamine, or sphingomyelin liposomes. PS and PI liposomes, but not the others, could effectively compete with PS liposomes and modified or native lipoproteins for binding to these receptors. Phosphatidic acid, another anionic phospholipid, could also compete, but was not as effective as PS or PI. Class B scavenger receptors are the first molecularly well-defined, specific cell surface receptors for anionic phospholipids to be described.