Scavenger receptor, class B, type I (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester without the uptake and degradation of the particle. In transfected cells SR-BI recognizes HDL, low density lipoprotein (LDL) and modified LDL, protein-free lipid vesicles containing anionic phospholipids, and recombinant lipoproteins containing apolipoprotein (apo) A-I, apoA-II, apoE, or apoCIII. The molecular basis for the recognition of such diverse ligands by SR-BI is unknown. We have used direct binding analysis and chemical cross-linking to examine the interaction of murine (m) SR-BI with apoA-I, the major protein of HDL. The results show that apoA-I in apoA-I/palmitoyl-oleoylphosphatidylcholine discs, HDL3, or in a lipid-free state binds to mSR-BI with high affinity (K(d) ≃ 5-8 μg/ml). ApoA-I in each of these forms was efficiently cross-linked to cell surface mSR-BI, indicating that direct protein-protein contacts are the predominant feature that drives the interaction between HDL and mSR-BI. When complexed with dimyristoylphosphatidylcholine, the N-terminal and C-terminal CNBr fragments of apoA-I each bound to SR-BI in a saturable, high affinity manner, and each cross-linked efficiently to mSR-BI. Thus, mSR-BI recognizes multiple sites in apoA-L A model class A amphipathic α-helix, 37pA, also showed high affinity binding and cross-linking to mSRBI. These studies identify the amphipathic α-helix as a recognition motif for SR-BI and lead to the hypothesis that mSR-BI interacts with HDL via the amphipathic α-helical repeat units of apoA-I. This hypothesis explains the interaction of SR-BI with a wide variety of apolipoproteins via a specific secondary structure, the class A amphipathic α-helix, that is a common structural motif in the apolipoproteins of HDL, as well as LDL.