The objective of this study was to search for brain-specific binding proteins that participated in Abeta aggregation. Immunoprecipitation of Abeta in Alzheimer's brain homogenate revealed a major co-precipitating 16-kDa protein band, which was identified through mass spectrometry as hemoglobin (Hb) alpha and beta chains. Hemoglobin was distributed in Alzheimer's disease (AD) patients in a brain region-dependent manner, with the highest levels in the hippocampus and parietal gray (PG) matter, followed by parietal white matter (PW), and the lowest in cerebellum (Cb). AD parietal gray and white matters exhibited higher Hb levels than those in the nondemented (ND) group. Likewise, RT-PCR revealed that the Hb mRNA levels in AD inferior temporal gyri were higher than those of ND subjects. Furthermore, Hb was shown to promote Abeta oligomer formation. Immunohistochemical studies indicated that Hb was localized within the cytosol of pyramidal neurons in the hippocampus, suggesting a potential source of intracerebral Hb. Finally, double immunofluorescent assay confirmed the co-localization of Hb with senile plaques (SP) and cerebral amyloid angiopathy (CAA). We propose that an elevation in brain Hb via circulation leakage or perturbations of Hb gene regulation may participate in AD pathogenesis.