We show that human and porcine polymorphonuclear leukocytes express significant amounts of cofilin, a low-molecular-weight actin regulatory protein, as well as profilin. Fifty percent of the cofilin in the resting state was phosphorylated and dephosphorylation occurred after activation by fMLP or TPA. The time course of the dephosphorylation induced by fMLP was very rapid, ending within 1 min, while TPA induced relatively gradual dephosphorylation over a period of 10 min. Surprisingly, okadaic acid and calyculin A, potent inhibitors specific for phosphatase, both induced dephosphorylation of cofilin. This suggests that type 1 alone or both type 1 and 2A phosphatases are involved in the maintenance of the level of phosphorylation of cofilin in resting cells. The dephosphorylation of cofilin was barely detected by in vitro phosphatase assays, which can distinguish activities of types 1, 2B, and 2C. This indicates that cofilin is not dephosphorylated by conventional phosphatases. Although the dephosphorylation of cofilin was observed in cells treated with the calcium ionophore A23187, the phosphorylation level of cofilin was restored when the cells were further incubated in the presence of EGTA. Reactivation of these cells with TPA resulted in the dephosphorylation of cofilin; fMLP activation did not lead to dephosphorylation. Furthermore, a submicromolar concentration of wortmannin, which is an inhibitor specific for phosphatidylinositol 3-kinase, completely inhibited dephosphorylation of cofilin induced by fMLP, but did not suppress TPA-induced dephosphorylation. Thus, we conclude that the dephosphorylation of cofilin is differently regulated depending on either fMLP or TPA activation.