The abundance and solubility of Aβ peptides are critical determinants of amyloidosis in Alzheimer's disease (AD). Hence, we compared levels of total soluble, insoluble, and total Aβ1-40 and Aβ1-42 in AD brains with those in age-matched normal and pathologic aging brains using a sandwich enzyme-linked immunosorbent assay (ELISA). Since the measurement of Aβ1-40 and Aβ1-42 depends critically on the specificity of the monoclonal antibodies used in the sandwich ELISA, we first demonstrated that each assay is specific for Aβ1-40 or Aβ1-42 and the levels of these peptides are not affected by the amyloid precursor protein in the brain extracts. Thus, this sandwich ELISA enabled us to show that the average levels of total cortical soluble and insoluble Aβ1-40 and Aβ1-42 were highest in AD, lowest in normal aging, and intermediate in pathologic aging. Remarkably, the average levels of insoluble Aβ1-40 were increased 20-fold while the average levels of insoluble Aβ1-42 were increased only 2-fold in the AD brains compared to pathologic aging brains. Further, the soluble pools of Aβ1-40 and Aβ1-42 were the largest fractions of total Aβ in the normal brain (i.e., 50 and 23%, respectively), but they were the smallest in the AD brain (i.e., 2.7 and 0.7%, respectively) and intermediate (i.e., 8 and 0.8%, respectively) in pathologic aging brains. Thus, our data suggest that pathologic aging is a transition state between normal aging and AD. More importantly, our findings imply that a progressive shift of brain Aβ1-40 and Aβ1-42 from soluble to insoluble pools and a profound increase in the levels of insoluble Aβ1-40 plays mechanistic roles in the onset and/or progression of AD.