The function of the neuronal microtubule-associated protein MAP1b is regulated by the degree of its phosphorylation, which is controlled in turn by activities of protein kinases and protein phosphatases (PP). To investigate the role of PP in the regulation of phosphorylation of MAP1b, okadaic acid and cyclosporin A were used to selectively inhibit PP2A and PP2B activities, respectively, in metabolically competent rat brain slices. The alterations of phosphorylation levels at mode Ⅰ sites of MAP1b were examined by Western blots using a phosphorylation-dependent MAP1b antibody 522 that specifically recognizes MAP1b phosphorylated at mode Ⅰ sites. The inhibition of PP2A, but not PP2B, was found to induce a marked increase in phosphorylation of MAP1b. Immunohistochemically, MAP1b was found ubiquitously in neuronal cell bodies and processes. A marked increase in neuronal staining in okadaic acid-treated tissue was observed with antibody 522 to the phosphorylated MAP1b. These results suggest that PP2A is the major PP that participates in regulation of the phosphorylation of MAP1b at the mode Ⅰ phosphorylation sites.