Phospholipase A2 (PLA2) and reactive oxygen species have been implicated both individually and synergistically in various forms of cellular injury. The form(s) of PLA2 important for cell injury and the implications of enhanced activity of the enzyme, however, have not been discerned. Previous studies reveal an increase in PLA2 activity associated with cell injury, but this association does not establish a causal relationship between the increase in activity and the injury. LLC-PK1 cell lines were created that express either the cytosolic PLA2 or a group II PLA2. The susceptibility of these cells to hydrogen peroxide toxicity was determined in order to evaluate the relative importance of these two forms of PLA2 in oxidant injury. Expression of cytosolic PLA2 in the LLC-cPLA2 cell line was associated with a 50-fold increase in PLA2 activity in the cytosolic fraction, an increase in agonist-stimulated arachidonate release, and immunodetection of the cytosolic PLA2 protein that was undetectable in control cells. Exposure to hydrogen peroxide or menadione, but not mercuric chloride, resulted in significantly greater lactate dehydrogenase release in LLC-cPLA2 cells when compared with control cells. Exogenous arachidonic acid (150 microM) did not enhance hydrogen peroxide-induced injury. The intracellular calcium chelator, 1,2-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/tetra(acetoxymethyl) ester, protected the cells against injury, but the calcium ionophore, A23187, did not increase injury. Glycine conferred no protective effect against hydrogen peroxide toxicity. By contrast to these results with cytosolic PLA2-expressing cells, secretory PLA2 expression to very high levels did not increase susceptibility to hydrogen peroxide. Thus, cytosolic PLA2 may an be an important mediator of oxidant damage to renal epithelial cells.