The double-stranded (ds) RNA-dependent protein kinase (PKR) is a primary regulator of antiviral responses; however, the ability of dsRNA to activate nuclear factor-κB (NF-κB) and dsRNA + interferon γ (IFN-γ) to stimulate inducible nitric-oxide synthase (iNOS) expression by macrophages isolated from PKR-/- mice suggests that signaling pathways in addition to PKR participate in antiviral activities. We have identified a novel phospholipid-signaling cascade that mediates macrophage activation by dsRNA and viral infection. Bromoenol lactone (BEL), a selective inhibitor of the calcium-independent phospholipase A2 (iPLA2), prevents dsRNA- and virus-induced iNOS expression by RAW 264.7 cells and mouse macrophages. BEL does not modulate dsRNA-induced interleukin 1 expression, nor does it affect dsRNA-induced NF-κB activation. Protein kinase A (PKA) and the cAMP response element binding protein (CREB) are downstream targets of iPLA2, because selective PKA inhibition prevents dsRNA-induced iNOS expression, and the inhibitory actions of BEL on dsRNA-induced iNOS expression are overcome by the direct activation of PKA. In addition, BEL inhibits dsRNA-induced CREB phosphorylation and CRE reporter activation. PKR does not participate in iPLA2 activation or iNOS expression, because dsRNA stimulates iPLA2 activity and dsRNA + IFN-γ induces iNOS expression and nitric oxide production to similar levels by macrophages isolated from PKR+/+ and PKR-/- mice. These findings support a PKR-independent signaling role for iPLA2 in the antiviral response of macrophages.