mRNA is an endogenous ligand for Toll-like receptor 3 (TLR3). This study demonstrates that RNA released from or associated with necrotic cells, as well as in vitro transcribed RNA, can activate TLR3 and induce immune responses. Human embryonic kidney 293 cells stably expressing TLR3 and containing a nuclear factor-κB (NF-κB)-dependent luciferase reporter were used to assess RNA-mediated TLR3 activation. Exposure to in vitro transcribed RNA resulted in TLR3-dependent induction of luciferase activity and interleukin-8 (IL-8) secretion. Treatment with in vitro transcribed mRNA activated NF-κB via TLR3 through a dose-dependent process involving tyrosine phosphorylation. In vitro transcribed natural or 2'-fluoro-substituted mRNA induced the expression of TLR3, interferon regulatory factor-1 (IRF-1), tumor necrosis factor-α (TNF-α), and interleukin-1 receptor-associated kinase-M (IRAK-M) mRNA in human dendritic cells (DCs). DCs responded to mRNA treatment by expressing activation markers, and this maturation was inhibited by an antagonistic TLR3-specific antibody. Endogenous RNA released from or associated with necrotic cells also stimulated DCs, leading to interferon-α (IFN-α) secretion, which could be abolished by pretreatment of necrotic cells with RNase. These results demonstrate that RNA, likely through secondary structure, is a potent host-derived activator of TLR3. This finding has potential physiologic relevance because RNA escaping from damaged tissue or contained within endocytosed cells could serve as an endogenous ligand for TLR3 that induces or otherwise modulates immune responses. The study also shows that mRNA can activate DCs, similar to the TLR3 ligand poly(I)-poly(C), and induces IRF-1, IRAK-M, TNF-α, and TLR3 mRNA accumulation. mRNA-triggered IL-8 secretion is tyrosine phosphorylation-dependent and PKR-independent. Necrotic cell-associated RNA induces IFN-α secretion by DCs. Antagonistic TLR3-specific antibody inhibits mRNA-induced DC activation. Dominant negative TRIF inhibits mRNA-induced IFN-β secretion. The study concludes that mRNA is a new endogenous ligand for TLR3 and demonstrates that RNA contained within necrotic cells activates DCs and alters their phenotype. This finding has implications for understanding the role of RNA in immune responses and the regulation of immune activation.mRNA is an endogenous ligand for Toll-like receptor 3 (TLR3). This study demonstrates that RNA released from or associated with necrotic cells, as well as in vitro transcribed RNA, can activate TLR3 and induce immune responses. Human embryonic kidney 293 cells stably expressing TLR3 and containing a nuclear factor-κB (NF-κB)-dependent luciferase reporter were used to assess RNA-mediated TLR3 activation. Exposure to in vitro transcribed RNA resulted in TLR3-dependent induction of luciferase activity and interleukin-8 (IL-8) secretion. Treatment with in vitro transcribed mRNA activated NF-κB via TLR3 through a dose-dependent process involving tyrosine phosphorylation. In vitro transcribed natural or 2'-fluoro-substituted mRNA induced the expression of TLR3, interferon regulatory factor-1 (IRF-1), tumor necrosis factor-α (TNF-α), and interleukin-1 receptor-associated kinase-M (IRAK-M) mRNA in human dendritic cells (DCs). DCs responded to mRNA treatment by expressing activation markers, and this maturation was inhibited by an antagonistic TLR3-specific antibody. Endogenous RNA released from or associated with necrotic cells also stimulated DCs, leading to interferon-α (IFN-α) secretion, which could be abolished by pretreatment of necrotic cells with RNase. These results demonstrate that RNA, likely through secondary structure, is a potent host-derived activator of TLR3. This finding has potential physiologic relevance because RNA escaping from damaged tissue or contained within endocytosed cells could serve as an endogenous ligand for TLR3 that induces or otherwise modulates immune responses. The study also shows that mRNA can activate DCs, similar to the TLR3 ligand poly(I)-poly(C), and induces IRF-1, IRAK-M, TNF-α, and TLR3 mRNA accumulation. mRNA-triggered IL-8 secretion is tyrosine phosphorylation-dependent and PKR-independent. Necrotic cell-associated RNA induces IFN-α secretion by DCs. Antagonistic TLR3-specific antibody inhibits mRNA-induced DC activation. Dominant negative TRIF inhibits mRNA-induced IFN-β secretion. The study concludes that mRNA is a new endogenous ligand for TLR3 and demonstrates that RNA contained within necrotic cells activates DCs and alters their phenotype. This finding has implications for understanding the role of RNA in immune responses and the regulation of immune activation.