Although CpG containing DNA is an important regulator of innate immune responses via toll-like receptor 9 (TLR9), excessive activation of this receptor is detrimental to the host. Here, we show that cytosolic phospholipase A, (cPLA(2)) activation is important for TLR9-mediated inducible nitric oxide synthase (iNOS) expression. Activation of TLR9 signaling by CpG induces iNOS expression and NO production. Inhibition of TLR9 blocked the iNOS expression and NO production. The CpG also stimulates cPLA(2)-hydrolyzed arachidonic acid (AA) release. Inhibition of cPLA2 activity by inhibitor attenuated the iNOS expression by CpG response. Additionally, knockdown of cPLA2 protein by miRNA also suppressed the CpG-induced iNOS expression. Furthermore, the CpG rapidly phosphorylates three MAPKs and Akt. A potent inhibitor for p38 MAPK or Akt blocked the CpG-induced AA release and iNOS expression. These results suggest that TLR9 activation stimulates cPLA(2) activity via p38 or Akt pathways and mediates iNOS expression. (C) 2007 Elsevier Inc. All rights reserved.