The chapter discusses the proteolytic processing of the β-amyloid precursor protein (APP) into amyloid β-peptide (Aβ), a major pathological hallmark of Alzheimer's disease. APP is a type-I transmembrane protein that undergoes sequential proteolytic processing by three secretase enzymes: α-, β-, and γ-secretase. The β- and γ-secretases generate Aβ, while α-secretase prevents Aβ generation by cleaving within the amyloid domain. The chapter outlines the cell biological and biochemical characteristics of these secretases and how APP matures and traffics through the secretory pathway to reach subcellular locations where they are active. It also highlights how neuronal activity and mutations causing familial Alzheimer's disease affect Aβ generation and disease progression. The text further details the roles of β-secretase (BACE1) and γ-secretase in Aβ production, including their intracellular trafficking and subcellular localization. Additionally, it explores the anti-amyloidogenic pathway mediated by α-secretase and the impact of familial Alzheimer's disease-associated mutations on Aβ generation and aggregation. The chapter concludes with insights into the subcellular sites of APP processing and the potential role of neuronal activity in regulating Aβ levels and metabolism.The chapter discusses the proteolytic processing of the β-amyloid precursor protein (APP) into amyloid β-peptide (Aβ), a major pathological hallmark of Alzheimer's disease. APP is a type-I transmembrane protein that undergoes sequential proteolytic processing by three secretase enzymes: α-, β-, and γ-secretase. The β- and γ-secretases generate Aβ, while α-secretase prevents Aβ generation by cleaving within the amyloid domain. The chapter outlines the cell biological and biochemical characteristics of these secretases and how APP matures and traffics through the secretory pathway to reach subcellular locations where they are active. It also highlights how neuronal activity and mutations causing familial Alzheimer's disease affect Aβ generation and disease progression. The text further details the roles of β-secretase (BACE1) and γ-secretase in Aβ production, including their intracellular trafficking and subcellular localization. Additionally, it explores the anti-amyloidogenic pathway mediated by α-secretase and the impact of familial Alzheimer's disease-associated mutations on Aβ generation and aggregation. The chapter concludes with insights into the subcellular sites of APP processing and the potential role of neuronal activity in regulating Aβ levels and metabolism.