Cofilin is an actin-depolymerizing protein, whose depolymerizing activity is supposed to be regulated in part by phosphorylation and dephosphorylation. Thus, we studied the phosphorylation states of cofilin in rat parotid acinar cells during stimulation for amylase exocytosis. Isoproterenol and carbachol induced rapid and extensive dephosphorylation of cofilin; 60-70% dephosphorylation was clearly detectable within 1min. Membranepermeable cyclic AMP (CPS-cAMP), phorbol ester (PMA), and Ca ionophore A 23187 mimicked the effect of isoproterenol and carbachol. Protein phosphatase inhibitors (calyculin A or FK 506 plus cyclosporin A) did not block the dephosphorylation in response to isoproterenol or carbachol. Furthermore, calyculin A alone strongly dephosphorylated cofilin. Although no exogenous protein phosphatases tested dephosphorylated cofilin in the homogenate, the cofilin that was isolated by immunoprecipitation was clearly dephosphorylated by protein phosphatases 1, 2 A, and 2 C.