IL-33, a member of the IL-1 family of cytokines, is produced by many cell types, including macrophages, yet its regulation is largely unknown. Treatment of primary murine macrophages with a panel of Toll-like receptor (TLR) (e.g., TLR2, TLR3, TLR4, and TLR9) agonists and non-TLR (e.g., MDA5, RIG-I) agonists revealed a pattern of gene and protein expression consistent with a role for IRF-3 in the expression of IL-33. Accordingly, induction of IL-33 mRNA was attenuated in IRF-3−/− macrophages and TBK1−/− mouse embryonic fibroblasts. Despite the fact that all IL-33 agonists were IRF-3-dependent, LPS-induced IL-33 mRNA was fully inducible in IFN-β−/− macrophages, indicating that IL-33 is not dependent on IFN-β as an intermediate. Epinephrine (EPI) and Bordetella pertussis adenylate cyclase toxin (ACT), cAMP-activating agents, activate CREB and greatly synergized with LPS to induce IL-33 mRNA in macrophages. Both LPS-induced and ACT/LPS-enhanced expression of IL-33 mRNA was partially, but significantly, inhibited by the Protein Kinase A (PKA) inhibitor, H-89, but not by tyrosine kinase or PKC inhibitors. Two IL-33 mRNA species derived from two alternative promoters encode full-length IL-33; however, the shorter “A” species is preferentially induced by all IL-33-inducing agonists except Newcastle Disease Virus (NDV), a RIG-I agonist, that induced expression of both “A” and “B” transcripts. Together, these studies greatly extend what is currently known about the regulation of IL-33 induction in macrophages stimulated by bacterial and viral agonists that engage distinct innate immune signaling pathways.