Cytosolic phospholipase A₂ (cPLA₂) is the rate-limiting key enzyme that cleaves arachidonic acid (AA) from membrane phospholipids for the biosynthesis of eicosanoids including prostaglandin E₂ (PGE₂), a key lipid mediator involved in inflammation and carcinogenesis. Here we show that cPLA₂ protein is S-nitrosylated and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric oxide synthase (iNOS) in human epithelial cells induced cPLA₂ S-nitrosylation, enhanced its catalytic activity and increased AA release. The iNOS-induced cPLA₂ activation is blocked by the specific iNOS inhibitor, 1400W. Addition of the NO donor, GSNO, to isolated cell lysates or purified recombinant human cPLA₂a protein induced S-nitrosylation of cPLA₂. Incubation of cultured cells with the iNOS substrate L-Arginine and NO donor significantly increased cPLA₂ activity and AA release. These findings demonstrate that iNOS-derived NO S-nitrosylates and activates cPLA₂ in human cells. Site-directed mutagenesis revealed that Cys-152 of cPLA₂ is critical for S-nitrosylation. Furthermore, COX-2 induction or expression markedly enhanced iNOS-induced cPLA₂ S-nitrosylation and activation, leading to 9, 23, 20 fold increase of AA release and 100, 38, 88 fold of PGE₂ production in A549, SG231, and HEK293 cells, respectively, whereas COX-2 alone leads to less than 2 fold change. These results indicate that COX-2 has the ability to enhance iNOS-induced cPLA₂ S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA₂ and COX-2. Since COX-2 enhances the formation of cPLA₂-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA₂ S-nitrosylation is mediated at least in part through increased association between iNOS and cPLA₂. These findings disclose a novel link among cPLA₂, iNOS and COX-2 which form a multiprotein complex leading to cPLA₂ S-nitrosylation and activation. Therefore, therapy aimed at disrupting this interplay may represent a promising strategy to effectively inhibit PGE₂ production and prevent inflammation and carcinogenesis.