Hydrogen peroxide (H(2)O(2)) stimulates the release of arachidonic acid from cells, but the signaling mechanism(s) involved remains to be elucidated. We investigated the roles of alpha-type cytosolic phospholipase A(2) (cPLA(2)alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of arachidonic acid from L929 cells (a murine fibroblast cell line), C12 cells (a variant of L929 that lacks cPLA(2)alpha) and a stable clone of C12 cells expressing cPLA(2)alpha (C12-cPLA(2)alpha cells). In the presence of 10 muM A23187, 100 nM phorbol myristate acetate (PMA) and 1 mM H(2)O(2) synergistically stimulated arachidonic acid release from L929 cells and C12-cPLA(2)alpha cells, and to a much lesser extent from C12 cells. The reagents alone and co-treatment with PMA and H(2)O(2) without A23187 had marginal effects. No arachidonic acid was released by PMA/A23187 or H(2)O(2)/A23187 in CaCl(2)-free buffer and the release was inhibited by a selective cPLA(2)alpha inhibitor (3 microM pyrrophenone). Addition of 10 microM H(2)O(2), which did not stimulate arachidonic acid release with A23187, enhanced the response to PMA/A23187. The release induced by PMA/A23187 and by H(2)O(2)/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X) and in PKC-depleted cells, and by a Src inhibitor (2 microM PP2). The phosphorylation of extracellular signal-regulated kinase 1/2 induced by PMA/A23187 and H(2)O(2)/A23187 was significantly decreased by inhibitors of PKC and Src. These findings suggest that H(2)O(2) with Ca(2+) stimulates arachidonic acid release via cPLA(2)alpha in a Src- and PKC-dependent manner in L929 cells. The role of cross-talk between Src and PKC in arachidonic acid release is discussed.