Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain. This hyperphosphorylated tau can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase-1, 2A, and 2B in vitro. In the present study, we have developed an assay to measure protein phosphatase activity toward tau-1 sites (Ser199/Ser202) using the hyperphosphorylated tau isolated from Alzheimer disease brain as substrate. Using this assay, we have identified that in normal brain, protein phosphatase-2A and 2B and, to a lesser extent, 1 are involved in the dephosphorylation of tau. The Km values of dephosphorylation of the hyperphosphorylated tau by protein phosphatase-2A and 2B are similar. The tau phosphatase activity is decreased by approximately 30% in brain of Alzheimer disease patients compared with those of age-matched controls. These findings suggest that a defect of protein phosphatase could be the cause of the abnormal hyperphosphorylation of tau in Alzheimer disease.