Exchangeable serum apolipoproteins and amphipathic alpha-helical peptides are effective inhibitors of sterol (free and esterified cholesterol) uptake at the small-intestinal brush border membrane. The minimal structural requirement of an inhibitor is an amphipathic alpha-helix of 18 amino acids. The inhibition is competitive, indicating that the inhibitor binds to scavenger receptor class B type I (SR-BI) present in the brush border membrane and responsible for sterol uptake. Binding of apolipoprotein A-I to SR-BI of rabbit brush border membrane is cooperative, characterized by a dissociation constant K(d) = 0.45 microM and a Hill coefficient of n = 2.8. The cooperativity of the interaction is due to binding of the inhibitor molecule to a dimeric or oligomeric form of SR-BI held together by disulfide bridges. Consistent with the competitive nature of the inhibition, the K(d) value agrees within experimental error with the IC(50) value of inhibition and with the inhibition constant K(I). After proteinase K treatment of brush border membrane vesicles, the affinity of the interaction of apolipoprotein A-I expressed as K(d) is reduced by a factor of 20, and the cooperativity is lost. The interaction of proteinase K-treated brush border membrane vesicles with apolipoprotein A-I is nonspecific partitioning of the apolipoprotein into the lipid bilayer of brush border membrane vesicles.