In rat uterine stromal cells (U-III cells), an oxidative stress induced by H2O2 caused a dose-dependent release of arachidonic acid (AA) that was independent of intracellular Ca2+ concentration and was not inhibited by Ca2+-dependent phospholipase A(2) (cPLA(2)) inhibitors, nor by protein kinase C (PKC) inhibitors or by PKC down-regulation. H2O2 treatment did not impair AA esterification but significantly increased Ca2+-independent PLA(2) (iPLA(2)) activity. Since iPLA(2) specific inhibitor bromoenollactone almost completely suppressed the release of AA induced by H2O2, we conclude that iPLA(2) activity represents the major mechanism by which H2O2 increases the availability of nonesterified AA in U-III cells. Moreover, PKC inhibitors sphingosine and calphostin C markedly potentiated the release of AA trigger by H2O2, suggesting a regulatory mechanism of iPLA(2) by PKC that remains to be clarified.