The phosphorylation sites on the human, 85-kDa cytosolic phospholipase A2 (cPLA2) were identified using recombinant cPLA2 expressed in Spodoptera frugiperda (Sf9) cells. Analysis by high performance liquid chromatography of tryptic digests of 32P-labeled recombinant cPLA2 showed four major peaks of radiolabeled phosphopeptides. The phosphorylated residues were identified as Ser-437, Ser-454, Ser-505, and Ser-727 using mass spectrometry and automated Edman sequencing. Sf9 cells infected with recombinant virus expressing cPLA2 exhibited a time-dependent release of arachidonic acid in response to the calcium ionophore A23187 or the protein phosphatase inhibitor okadaic acid, which was not observed in Sf9 cells infected with wild-type virus. Stimulation of Sf9 cells with A23187 and okadaic acid also increased the level of phosphorylation of cPLA2. Okadaic acid, but not A23187, induced a gel shift of cPLA2 and increased the level of phosphorylation of Ser-727 by 4.5-fold, whereas the level of phosphorylation of the other sites increased by 60% or less in response to both agonists. To determine whether the same sites on cPLA2 were phosphorylated in mammalian cells, human monocytes were studied. Okadaic acid stimulation of monocytes induced a gel shift of cPLA2, increased the release of arachidonic acid, and increased the level of phosphorylation of cPLA2 on serine residues. Comparison of two-dimensional peptide maps of tryptic digests of 32P-labeled recombinant cPLA2 and human monocyte cPLA2 demonstrated that the same peptides on cPLA2 were phosphorylated in mammalian cells as in insect cells. These results show that the Sf9-baculovirus expression system is useful for investigation of the phosphorylation sites on cPLA2. The results also suggest that phosphorylation of the cPLA2 by protein kinases other than mitogen-activated protein kinase may be important for the regulation of arachidonic acid release.