There is accumulating evidence that soluble amyloid-beta (Abeta) oligomers, rather than amyloid fibrils, are the principal pathogenic species in Alzheimer disease (AD). Here, we have developed a novel enzyme-linked immunosorbent assay (ELISA) specific for high-molecular-weight (HMW) Abeta oligomers. Analysis of Abeta oligomers derived from synthetic Abeta 1-42, by size-exclusion chromatography (SEC), revealed that our ELISA specifically detected HMW Abeta oligomers of 40-200 kDa. Using this ELISA, we detected significantly higher (P<0.0001) signals in cerebrospinal fluid (CSF) samples from 25 patients with AD or mild cognitive impairment (MCI), compared to 25 age-matched controls. As a test for discriminating between the AD/MCI and control groups, the area under the curve in receiver operating characteristic analysis for the CSF HMW Abeta oligomers was greater than that for CSF Abeta x-42. Furthermore, the CSF levels of HMW Abeta oligomers showed a negative correlation with Mini-Mental State Examination scores in the AD/MCI group. We conclude that the CSF HMW Abeta oligomers detected by our ELISA could be useful as a diagnostic marker for AD, and also as a potential surrogate marker for disease severity. Our results support the idea that soluble HMW Abeta oligomers play a critical role in the pathogenesis and progression of AD.