Early endosomes are a major site of amyloid precursor protein (APP) processing and a convergence point for molecules of pathologic relevance to Alzheimer’s disease (AD). Neuronal endosome enlargement, reflecting altered endocytic function, is a disease-specific response that develops years before the earliest stage of AD and Down syndrome (DS). We examined how endocytic dysfunction is related to Aβ accumulation and distribution in early stage AD and DS. We found by ELISA and immunocytochemistry that the appearance of enlarged endosomes coincided with an initial rise in soluble Aβ40 and Aβ42 peptides, which preceded amyloid deposition. Double-immunofluorescence using numerous Aβ antibodies showed that intracellular Aβ localized principally to rab5-positive endosomes in neurons from AD brains and was prominent in enlarged endosomes. Aβ was not detectable in neurons from normal controls and was diminished after amyloid deposition in neuropathologically confirmed AD. These studies support growing evidence that endosomal pathology contributes significantly to Aβ overproduction and accumulation in sporadic AD and in AD associated with DS and may signify earlier disease-relevant disturbances of the signaling functions of endosomes.