Tau hyperphosphorylation leads to formation of paired helical filament/neurofibrillary tangles, the hallmark lesion seen in Alzheimer's disease (AD) brain. An imbalanced regulation in protein kinases and protein phosphatases in the affected neurons is proposed to be a reasonable causative factor to the disease process. To verify the hypothesis, we have injected in the present study calyculin A, a potent and specific inhibitor of protein phosphatase (PP) 2A and PP1, into rat hippocampus bilaterally, thus reproduced an Alzheimer's-like deficiency in dephosphorylation system. It was found that calyculin A-injected rats developed lesions in spatial memory retention in Morris water maze test. At mean time, tau was hyperphosphorylated at Ser396/Ser404 (PHF-1) and Ser-262/Ser-356 (12E8) sites determined both by immunohistochemistry and Western blot. It is implicated that (1) PP2A and PP1 participate in the in vivo regulation of tau phosphorylation, and down-regulation of the two phosphatases will result in tau hyperphosphorylation; (2) hyperphosphorylation of tau at PHF-1 and 12E8 sites might be crucial to affect spatial memory in AD.