Lipid rafts regulate the amyloidogenic processing of the Alzheimer's β-amyloid precursor protein (APP). This study demonstrates that cholesterol levels significantly influence the production of β-amyloid (Aβ) peptides derived from APP. Reducing cholesterol in N2a cells decreased Aβ production, while increasing cholesterol levels enhanced it. APP and BACE1, a β-secretase, were found to associate with lipid rafts, which are cholesterol- and sphingolipid-rich microdomains. Antibody cross-linking increased the association of APP and BACE1 with detergent-resistant membranes (DRMs), indicating their localization in lipid rafts. This association is critical for β-secretase activity, which cleaves APP to generate Aβ. In contrast, α-secretase cleavage, which prevents Aβ formation, occurs outside of lipid rafts. The study also shows that endocytosis is essential for β-cleavage, as inhibiting endocytosis reduced Aβ production. However, antibody-induced cross-linking of APP and BACE1 in lipid rafts could overcome this inhibition, leading to increased Aβ production. These findings suggest that lipid rafts regulate the access of secretases to APP, thereby controlling Aβ generation. The study highlights the importance of lipid rafts in the pathogenesis of Alzheimer's disease by influencing the processing of APP and the formation of amyloid plaques.Lipid rafts regulate the amyloidogenic processing of the Alzheimer's β-amyloid precursor protein (APP). This study demonstrates that cholesterol levels significantly influence the production of β-amyloid (Aβ) peptides derived from APP. Reducing cholesterol in N2a cells decreased Aβ production, while increasing cholesterol levels enhanced it. APP and BACE1, a β-secretase, were found to associate with lipid rafts, which are cholesterol- and sphingolipid-rich microdomains. Antibody cross-linking increased the association of APP and BACE1 with detergent-resistant membranes (DRMs), indicating their localization in lipid rafts. This association is critical for β-secretase activity, which cleaves APP to generate Aβ. In contrast, α-secretase cleavage, which prevents Aβ formation, occurs outside of lipid rafts. The study also shows that endocytosis is essential for β-cleavage, as inhibiting endocytosis reduced Aβ production. However, antibody-induced cross-linking of APP and BACE1 in lipid rafts could overcome this inhibition, leading to increased Aβ production. These findings suggest that lipid rafts regulate the access of secretases to APP, thereby controlling Aβ generation. The study highlights the importance of lipid rafts in the pathogenesis of Alzheimer's disease by influencing the processing of APP and the formation of amyloid plaques.