γ-Secretase is a proteolytic complex whose substrates include Notch, β-amyloid precursor protein (APP), and several other type I transmembrane proteins. Presenilin (PS) and nicastrin are known components of this high-molecular-weight complex, and recent genetic screens in invertebrates have identified two additional gene products, Aph1 and Pen-2, as key factors in γ-secretase activity. Here, we examined the interaction of the components of the γ-secretase complex in Chinese hamster ovary cells stably expressing human forms of APP, PS1, Aph1, and Pen-2. Subcellular fractionation of membrane vesicles and subsequent coimmunoprecipitation of individual γ-secretase components revealed that interactions among all proteins occurred in the Golgi/trans-Golgi network (TGN) compartments. Furthermore, incubation of the Golgi/TGN-enriched vesicles resulted in de novo generation of amyloid β-protein and APP intracellular domain. Immunofluorescent staining of the individual γ-secretase components supported our biochemical evidence that the γ-secretase components assemble into the proteolytically active γ-secretase complex in the Golgi/TGN compartment.