Recent evidence supports a regulatory role for the calcium-independent phospholipase A2 (iPLA2) in the antiviral response of inducible nitric-oxide synthase (iNOS) expression by macrophages. Because two mammalian isoforms of iPLA2 (iPLA2beta and iPLA2gamma) have been cloned and characterized, the aim of this study was to identify the specific isoform(s) in macrophages that regulates the expression of iNOS in response to virus infection. Bromoenol lactone (BEL), a suicide substrate inhibitor of iPLA2, inhibits the activity of both isoforms at low micromolar concentrations. However, the R- and S-enantiomers of BEL display approximately 10-fold greater potency for inhibition of the enzymatic activity of iPLA2gamma and iPLA2beta, respectively. In this study, we show that the iPLA2beta-selective (S)-BEL inhibits encephalomyocarditis virus (EMCV)-induced iNOS expression, nitric oxide production, and iPLA2 enzymatic activity in macrophages in a concentration-related manner that closely resembles the inhibitory properties of racemic BEL. cAMP response element-binding protein (CREB) is one downstream target of iPLA2 that is required for the transcriptional activation of iNOS in response to virus infection, and consistent with the effects of BEL enantiomers on iNOS expression, (S)-BEL more effectively inhibits EMCV-induced CREB phosphorylation than (R)-BEL in macrophages. Using macrophages isolated from iPLA2beta-null mice, virus infection fails to stimulate iNOS mRNA accumulation and protein expression, thus providing genetic evidence that iPLA2beta is required for EMCV-induced iNOS expression. These findings provide evidence for a signaling role for iPLA2beta in virus-induced iNOS expression by macrophages.