gamma-Secretase is a multi-subunit membrane protease complex that catalyses the final intramembrane cleavage of the beta-amyloid precursor protein (APP) during the neuronal production of amyloid-beta peptides (A beta), which are implicated as the causative agents of Alzheimer's disease (AD). In the present study, we report the reconstitution of a highly purified, active gamma-secretase complex into proteoliposomes without exogenous lipids and provide the first direct evidence for the existence of a microenvironment of 53 molecular species from 11 major lipid classes specifically associated with the gamma-secretase complex, including phosphatidylcholine and cholesterol. Importantly, we demonstrate that the pharmacological modulation of certain phospholipids abolishes both the integrity and the enzymatic activity of the intramembrane protease. Together, our findings highlight the importance of a specific lipid microenvironment for the structure and function of gamma-secretase.