Protein phosphatase 2A and GSK–3β are important factors involved in tau hyperphosphorylation. PP–2A is able to dephosphorylate GSK–3β in vitro and has been proposed to regulate GSK–3β activity in vivo. From the finding that lithium can block ceramide induced activation of a protein phosphatase with characteristics similar to PP–2A, it could imply that GSK–3 regulates PP–2A in a manner similar to the regulation of PP–1. The mechanism of glycogen synthase kinase–3β regulating protein phosphatase 2A's activity was investigated. GSK–3β was found to be co–precipitated with I2PP–2A. I2PP–2A and PP–2A C subunit protein levels were detected by Western blots after inhibition or activation of GSK–3β. I2PP–2A immunoreactivity was decreased while treated with LiCl and SB216763, two GSK–3 inhibitors. And PP–2A C subunit protein level was decreased after treated with LiCl for 24 hours, but increased after treated with SB216763 for one hour. On the contrary, while increase activity of GSK–3β by wortmannin for one hour or transient transfection with GSK–3β plasmid, I2PP–2A protein level was found to decrease and PP–2A C subunit protein level was increased. These results indicated that I2PP–2A could form a stable complex with GSK–3β in vivo. And GSK–3β may possibly regulate PP–2A activity by changing the expression of PP–2A C subunit protein and I2PP–2A protein.