The 85-kDa cytosolic PLA₂ (CPLA₂) mediates agonist-induced arachidonic acid release in many cell models, including mouse peritoneal macrophages. cPLA₂ is regulated by an increase in intracellular calcium, which binds to an amino- terminal C2 domain and induces its translocation to the nuclear envelope and endoplasmic reticulum. Phosphorylation of cPLA₂ on S505 by mitogen-activated protein kinases (MAPK) also contributes to activation. In macrophages, zymosan induces a transient increase in intracellular calcium and activation of MAPK, which together fully activate cPLA₂ and synergistically promote arachidonic acid release. There are alternative pathways for regulating cPLA₂ in macrophages because PMA and okadaic acid induce arachidonic acid release without increasing calcium. The baculovirus expression system is a useful model to study cPLA₂ activation. Sf9 cells expressing cPLA₂ release arachidonic acid to either A23187 or okadaic acid. cPLA₂ is phosphorylated on multiple sites in Sf9 cells, and phosphorylation of S727 is preferentially induced by okadaic acid. However, the phosphorylation sites are non-essential and only S505 phosphorylation partially contributes to cPLA₂ activation in this model. Although okadaic acid does not increase intracellular calcium in Sf9 cells, calcium binding by the C2 domain is necessary for arachidonic acid release. A23187 and okadaic acid activate cPLA₂ by different mechanisms, yet both induce translocation to the nuclear envelope in Sf9 cells. The results demonstrate that alternative regulatory pathways can lead to cPLA₂ activation and arachidonic acid release. J. Leukoc. Biol. 65: 330–336; 1999.