The disruption of intracellular calcium homeostasis plays a central role in the pathology of Alzheimer's disease, which is also characterized by accumulation of the amyloid-β peptides Aβ40 and Aβ42. These amphipathic peptides may become associated with neuronal membranes and affect their barrier function, resulting in the loss of calcium homeostasis. This suggestion has been extensively investigated by exposing protein-free model membranes, either vesicles or planar bilayers, to soluble Aβ. Primarily unstructured Aβ has been shown to undergo a membrane-induced conformational change to either primarily β-structure or helical structure, depending, among other factors, on the model membrane composition. Association of Aβ renders lipid bilayers permeable to ions but there is dispute whether this is due to the formation of discrete transmembrane ion channels of Aβ peptides, or to a non-specific perturbation of bilayer integrity by lipid head group-associated Aβ. Here, we have attempted incorporation of Aβ in the hydrophobic core of zwitterionic bilayers, the most simple model membrane system, by preparing proteoliposomes by hydration of a mixed film of Aβ peptides and phosphatidylcholine (PC) lipids. Despite the use of a solvent mixture in which Aβ40 and Aβ42 are almost entirely helical, the Aβ analogs were β-structured in the resulting vesicle dispersions. When Aβ40-containing vesicles were fused into a zwitterionic planar bilayer, the typical irregular “single channel-like” conductance of Aβ was observed. The maximum conductance increased with additional vesicle fusion, while still exhibiting single channel-like behavior. Supported bilayers formed from Aβ40/PC vesicles did not exhibit any channel-like topological features, but the bilayer destabilized in time. Aβ40 was present primarily as β-sheets in supported multilayers formed from the same vesicles. The combined observations argue for a non-specific perturbation of zwitterionic bilayers by surface association of small amphipathic Aβ40 assemblies.