An early event in Alzheimer's disease (AD) pathogenesis is the formation of extracellular aggregates of amyloid-β peptide (Aβ), thought to be initiated by a prion-like seeding mechanism. However, the molecular nature and location of the Aβ seeds remain rather elusive. Active Aβ seeds are found in crude homogenates of amyloid-laden brains and in the soluble fraction thereof. To analyze the seeding activity of the pellet fraction, we have either separated or directly immunoisolated membranes from such homogenates. Here, we found considerable Aβ seeding activity associated with membranes in the absence of detectable amyloid fibrils. We also found that Aβ seeds on mitochondrial or associated membranes efficiently induced Aβ aggregation in vitro and seed β-amyloidosis in vivo. Aβ seeds at intracellular membranes may contribute to the spreading of Aβ aggregation along neuronal pathways and to the induction of intracellular pathologies downstream of Aβ.