In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to morphological changes in GEC and proteinuria, in association with phospholipase A₂ (PLA₂) activation. The present study addresses the role of calcium-independent PLA₂ (iPLA₂) in GEC injury. iPLA₂-short and iPLA₂ were expressed in cultured rat GEC and normal rat glomeruli. To determine if iPLA₂ is involved in complement-mediated arachidonic acid (AA) release, GEC were stably transfected with iPLA₂ or iPLA₂ cDNAs (GEC-iPLA₂; GEC-iPLA₂). Compared with control cells (GEC-Neo), the GEC-iPLA₂ and GEC-iPLA₂ demonstrated greater expression of iPLA₂ proteins and activities. Complement-mediated release of [³H]AA was augmented significantly in GEC-iPLA₂, as compared with GEC-Neo, and the augmented [³H]AA release was inhibited by the iPLA₂-directed inhibitor bromoenol lactone (BEL). For comparison, overexpression of iPLA₂ also amplified [³H]AA release after incubation of GEC with H₂O₂, or chemical anoxia followed by re-exposure to glucose (in vitro ischemia-reperfusion injury). In parallel with release of [³H]AA, complement-mediated production of prostaglandin E₂ was amplified in GEC-iPLA₂. Complement-mediated cytotoxicity was attenuated significantly in GEC-iPLA₂, as compared with GEC-Neo, and the cytoprotective effect of iPLA₂ was reversed by BEL, and in part by indomethacin. Overexpression of iPLA₂ did not amplify complement-dependent [³H]AA release, but nonetheless attenuated complement-mediated cytotoxicity. Thus, iPLA₂. may be involved in complement-mediated release of AA. Expression of iPLA₂ or iPLA₂ induces cytoprotection against complement-dependent GEC injury. Modulation of iPLA₂ activity may prove to be a novel approach to reducing GEC injury.