Abstract: One of the major pathological features of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques that are predominantly composed of the amyloid-β peptide (Aβ). Characterization of plaques demonstrated the predominance of two peptides differing at the carboxyl terminus by two hydrophobic amino acids, Aβ40 and Aβ42. Diffuse plaques associated with AD are composed predominantly of Aβ42, whereas senile plaques contain both Aβ40 and Aβ42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane-bound Aβ species and that Aβ42 is the critical component. In order to investigate this hypothesis, we have examined Aβ40/42-lipid interactions using in situ atomic force microscopy, electron microscopy, and fluorescence anisotropy. While the association of Aβ42 with planar bilayers resulted in peptide aggregation, but no fiber formation, this was not the case for Aβ40, where we observed preferential fiber formation. Cholesterol, a key membrane component and modulating factor in AD, is inversely correlated with the extent of Aβ40/42-bilayer interaction. These results were confirmed using fluorescence anisotropy to evaluate the effect of Aβ on membrane fluidity and fluorimetry to confirm membrane integrity. Our results suggest that the enhanced amyloidogenic properties of Aβ42 are not correlated with fibril formation, but with aggregation on bilayer surfaces.