Theiler's murine encephalomyelitis virus (TMEV) infection in the central nervous system (CNS) induces a demyelinating disease similar to human multiple sclerosis. TMEV infection results in activation of various chemokine and cytokine genes that are important in the initiation of an inflammatory response. We have previously shown that the production of these chemokines and cytokines in astrocytes is induced via the NF-kappaB pathway following TMEV and Coxsackie virus infection. In this study, we investigated whether the NF-kappaB-dependent inflammatory responses after TMEV infection is triggered through TLR3 and/or TLR7. The activation of NF-kappaB or IRF/ISRE, as well as the production of both MCP-1/CCL2 and IL-8/CXCL8, was observed in only TLR3-transfected HEK 293 cells, but not in TLR7-tranfected cells. The potential involvement of TLR3 in mouse embryonic fibroblasts and primary astrocytes was further investigated following transfection with wildtype or dominant negative form of TLRs and MyD88, as well as astrocytes from TLR3- and MyD88-deficient mice. Similarly, the activation of transcription factors and chemokine genes is induced in these mouse cells through primarily TLR3 signaling pathway, but not TLR7 or other MyD88-mediated pathways following TMEV infection. However, the TLR3-mediated cellular activation does not appear to affect the level of viral replication in astrocytes. These results strongly suggest that TLR3-signaling by TMEV alone is sufficient to induce the initial inflammatory cytokine responses that could be very important for the outcome of virus-induced encephalitis and/or demyelinating diseases, such as multiple sclerosis.