In the present study, we have assessed the biophysical properties of lipid rafts from different brain areas in subjects exhibiting early neuropathological stages of Alzheimer's disease (AD). By means of steady-state fluorescence polarization analyses using two environment-sensitive fluorescent probes, we demonstrate that lipid rafts from cerebellum, and frontal and entorhinal cortices, exhibit different biophysical behaviors depending on the stage of the disease. Thus, while membrane anisotropies were similar in the cerebellum along stages, lipid rafts from frontal and entorhinal cortices at AD stages I/II and AD III were significantly more liquid-ordered than in control subjects, both at the aqueous interface and hydrophobic core of the raft membrane. Thermotropic analyses demonstrated the presence of Arrhenius breakpoints between 28.3-32.0 °C, which were not influenced by the disease stage. However, analyses of membrane microviscosity (ηapp) demonstrate that frontal and entorhinal lipid rafts are notably more viscous and liquid-ordered all across the membrane from early stages of the disease. These physicochemical alterations in lipid rafts do not correlate with changes in cholesterol or sphingomyelin levels, but to reduced unsaturation index and increased saturate/polyunsaturated ratios in phospholipid acyl chains. Moreover, we demonstrate that β-secretase/AβPP (amyloid-β protein precursor) interaction and lipid raft microviscosity are strongly, and positively, correlated in AD frontal and entorhinal cortices. These observations strengthens the hypothesis that physical properties of these microdomains modulate the convergence of amyloidogenic machinery toward lipid rafts, and also points to a critical role of polyunsaturated fatty acids in amyloidogenic processing of AβPP.