Failure in amyloid-β (Aβ) systemic clearance across the liver has been suggested to play a role in Aβ brain accumulation and thus to contribute largely to the pathology of Alzheimer's disease (AD). The purpose of this study was to characterize in vitro the transport mechanisms of Aβ₄₀ across the liver using sandwich-cultured primary rat hepatocytes (SCHs) and to determine its biliary clearance (CL(bile)) and biliary excretion index (BEI%). ¹²⁵I-Aβ₄₀ BEI% was time dependent and reached steady state at 30 minutes, with an average value of 29.8% and a CL(bile) of 1.47 ml/min per kilogram of body weight. The role of low-density lipoprotein receptor-related protein-1 (LRP1) in mediating the basolateral uptake of ¹²⁵I-Aβ₄₀ in SCHs was assessed using receptor-associated protein (RAP, 2 µM). A significant reduction in ¹²⁵I-Aβ₄₀ BEI% and CL(bile) with RAP was observed, demonstrating a major contribution of LRP1 in mediating hepatic uptake of intact ¹²⁵I-Aβ₄₀ via transcytosis. Furthermore, activity studies suggested a lower role of receptor for advanced glycation end products (RAGE) in ¹²⁵I-Aβ₄₀ hepatic uptake. Verapamil (50 µM) and valspodar (20 µM) significantly reduced ¹²⁵I-Aβ₄₀ BEI%, indicating a role for P-glycoprotein (P-gp) in the biliary excretion of ¹²⁵I-Aβ₄₀ in SCHs. LRP1- and P-gp-mediated ¹²⁵I-Aβ₄₀ biliary excretion was inducible and increased BEI% by 26% after rifampicin pretreatment. In conclusion, our findings demonstrated that besides LRP1, P-gp and, to a lesser extent, RAGE are involved in ¹²⁵I-Aβ₄₀ hepatobiliary disposition and support the use of enhancement of Aβ hepatic clearance via LRP1 and P-gp induction as a novel therapeutic approach for the prevention and treatment of AD.