The high density lipoprotein receptor, scavenger receptor class B type I (SR-BI), recognizes lipid-bound apolipoprotein A-I (apoA-I) and other apolipoproteins. Here, we have used large scale cultures of apoE-expressing cells to purify apoE and prepare apoE containing reconstituted discoidal 1-palmitoyl-2-oleoyl-l-phosphatidylcholine (POPC)-apoE particles. These particles have been used to examine their binding to wild-type and mutant forms of SR-BI expressed in transfected ldlA-7 cells. Specific binding to SR-BI was determined by subtracting from the total binding, nonspecific values measured using either control untransfected ldlA-7 cells or by inhibiting SR-BI-mediated binding with a high titer antireceptor-blocking antibody. POPC-apoE particles generated using apoE2, apoE3, apoE4, or the carboxyl-terminally truncated forms apoE165, apoE202, apoE229, and apoE259 all bound tightly to wild-type SR-BI with similar affinities (K(d) = 35-45 microg/ml). Binding was nearly abolished in a cell line expressing the ldlA (Q402R/Q418R) double mutant form of SR-BI that is unable to bind native high density lipoprotein but binds low density lipoprotein normally. The findings establish that apoE is a ligand for SR-BI and that the receptor binding domain is located in the amino-terminal 1-165-region of the protein. SR-BI-apoE interactions may contribute to cholesterol homeostasis in tissues and cells expressing SR-BI that are accessible to apoE-containing lipoproteins.