Proteins binding to amyloid beta-protein (Abeta) may modulate the accumulation of Abeta in Alzheimer's disease (AD) brain. We developed a monomeric Abeta column for isolation of the proteins binding to Abeta from rat brain. By amino acid sequence analysis and immunoreactivity with specific antibodies, we identified three new Abeta-binding proteins, glutamine synthetase, hemoglobin alpha-chain, and macrophage migration inhibitory factor as well as serum albumin, beta-tubulin, and glyceraldehyde-3-phosphate dehydrogenase already identified as proteins bound to amyloid beta-protein precursor. In addition, the retained fraction contained both apolipoprotein E and alpha(1)-antichymotrypsin already known as Abeta binding proteins. Furthermore, we detected the complexes of these new binding proteins with Abeta in a soluble fraction of the cerebral cortex of AD brain by immunoprecipitation. Our results suggest that these binding proteins also associate with Abeta, leading to the clearance or the accumulation of Abeta and the neuronal cell damage in human brain.