Cytosolic phospholipase A₂ (cPLA₂) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA₂ is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA₂ were investigated. In Sf9 insect cells expressing cPLA₂, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA₂ to the nuclear envelope. cPLA₂ is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA₂ activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA₂, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA₂ linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA₂ to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.